17α-monoesters and 17α,21-diesters of cortexolone for use in the treatment of tumors

ABSTRACT

The present invention relates to certain cortexolone derivatives of formula (I) 
                         
and the use of the same as antitumor active ingredients for the curative or adjuvant, or neoadjuvant or palliative treatment of precancerous lesions, dysplasias, metaplasias and tumor diseases, including malignant neoplasias and metastasis. The present invention also relates to pharmaceutical compositions comprising cortexolone derivatives of formula (I) as active ingredients and at least one physiologically acceptable excipient, and to the use of the pharmaceutical compositions as antitumor medicinal products.

CROSS REFERENCE TO RELATED APPLICATIONS

The instant application is a division of U.S. patent application Ser.No. 15/517,653, filed on 7 Apr. 2017, which in turn is is a nationalstage filing under 35 U.S.C. § 371 of PCT/EP2015/073172, filed on 7 Oct.2015, and claims the benefit of priority to European Patent ApplicationNo. 14188063.3, filed 8 Oct. 2014. Each application is incorporatedherein by reference in its entirety.

SUMMARY OF THE INVENTION

The present invention provides certain cortexolone derivatives offormula (I):

and the same for use as antitumor active ingredients for the curative oradjuvant, or neoadjuvant or palliative treatment of precancerouslesions, dysplasias, metaplasias and tumor diseases, including malignantneoplasias and metastasis.

Another aspect of the invention relates to pharmaceutical compositionscomprising at least one cortexolone derivative of formula (I) as activeingredient with at least one physiologically acceptable excipient, andto the same pharmaceutical compositions for use as antitumor medicinalproducts for the curative or adjuvant, or neoadjuvant or palliativetreatment of precancerous lesions, dysplasias, metaplasias and tumordiseases, including malignant neoplasias and metastasis.

BACKGROUND OF THE INVENTION

Tumor, or neoplasm, is defined as a mass of new tissue which persistsand grows independently of its surrounding structures, and which has nophysiological use (Doreland's Medical Dictionary, 23 ED. 1960).

Several classifications are available for tumors: for the exploitationof this patent application, the most important are the epithelialtumors.

The epithelial tumors are neoplasms derived from epithelial cells, thetype of cell which lines hollow internal organs and body surfaces; thisgroup includes many of the most common cancers, and includes most ofthose developing in the breast, prostate, lung, pancreas, andgastrointestinal tract.

In some cases, the epithelial tumors can also be characterized by thepresence of specific hormone-receptors in the tumor cells which gives tothe tumor a hormone-sensitivity.

Carcinomas, that are malignant tumors derived from epithelial cells,make up about 85 out of every 100 cancers (85%).

One example of epithelial carcinoma is the pancreatic carcinoma (alsoreferred to as pancreatic cancer).

Pancreatic cancer is one of the most deadly forms of carcinomas. Theexocrine and endocrine cells of the pancreas form completely differenttypes of tumors. Exocrine pancreatic tumors constitute the most commontype of pancreatic cancer (more than 95%). Although benign(non-cancerous) cysts and benign tumors (adenomas) may develop in thepancreas, most of the exocrine pancreatic tumors are malignant.

The carcinoma of pancreas, particularly exocrine pancreas carcinoma andmuch more particularly the most frequent one, that is ductaladenocarcinoma, falls into the five most frequent causes of death inmales, and is the fourth cause of death in females. It is one of thetumors with the highest unfavorable prognosis, with a survival of only5% in males and 6% in the females at 5 years after diagnosis. Thehighest incidence occurs between 60-70 years of age (AIOM. Linea GuidaCarcinoma del Pancreas Esocrino, ed. 2013).

The etiology of the exocrine pancreas carcinoma is unknown. There is arecognized genetic predisposition (familiarity) and some risk factorssuch as smoke, fatty diet, diabetes mellitus type 2, chronicpancreatitis, environmental factors such as solvents or pesticides.

The carcinoma of the exocrine pancreas is, in its early stage,asymptomatic, and this explains the delay in the diagnosis, which isusually performed when the disease is at an advanced stage, withexception for accidental detection during diagnostic procedures forother abdominal diseases.

Patients diagnosed with pancreatic cancer typically have a poorprognosis: considering the above described delay in the diagnosis, onlyabout 15% of cases show the tumor limited to the pancreas, whereas inthe remaining cases, the diffusion to the loco-regional lymphnodes isdetected in about 25% of the patients, and the presence of metastases isdetected in 60% of the cases.

Median survival from diagnosis of the cancer is approximately three tosix months, while a five-year survival is significantly less than 5%.

The therapy of carcinoma of the pancreas is surgery, when possible, alsowith palliative purposes.

Radical pancreaticoduodenectomy is currently the only chance of cure,especially for minimal disease.

The medical therapy, also associated to radiotherapy, is limited to theunresectable cases, or when metastases are present, or as adjuvanttreatment after surgery. Although there are occasional reports ofindividual patients who respond to gemcitabine or fluorouracil, orcombination regimens with doxorubicin, methotrexate, cisplatin,oxaliplatin, irinotecan, erlotinib and so on, the results ofchemotherapy are generally unsatisfactory and often no better than notreatment at all (Martindale, 31 ed., page 530).

Theve et al, in 1983 reviewed possible effects of sex hormones on thepancreas, based on reports on steroid receptor proteins in pancreatictissue, the high capacity of estrogen binding protein in the humanpancreas and capacity of human pancreatic tissue to convert the mainperipheral estrogen, estrone sulphate, into the terminal biologicallyactive estradiol-17 beta.

With this background, they tried tamoxifen (an antagonist of theestrogen receptor) in patients with unresectable adenocarcinoma of thepancreas with some preliminary results similar to those by Wong et al.,in 1993.

The clinical practice in the subsequent years did not give the expectedresults, but the conclusion was that even if anti-estrogens did notconstitute the optimal form of therapy, other sorts of hormonalmanipulation ought to be tried in pancreatic cancer.

In view of the above, there is a strong need for new approaches of tumortreatment and, in particular, for the treatment of carcinomas, and stillmore especially for the treatment of epithelial tumors, especiallyprostatic carcinoma or pancreas carcinoma (preferably exocrine pancreascarcinoma).

A number of compounds referred to as 17α-monoesters, 21-monoesters and17α,21-diesters of cortexolone and processes for their manufacturing areknown in the art.

WO03/014141 describes compounds belonging to the family of steroidsstructurally related to cortexolone (also known as 11-deoxycortisone) ashaving mainly antiandrogenic activity. These compounds, such ascortexolone 17α-propionate, act by interfering with the direct action ofthe androgenic hormones on the Androgen Receptor (AR) in the tissues.

WO2007/031349 discloses C₃-C₁₀ 17α-esters of 9,11-dehydrocortexolone, aderivative structurally related to cortexolone, as antigonadotrophicagent, which may be useful for the treatment disorders closely relatedto excess of gonadotrophin production.

WO2009/019138 discloses an enzymatic process for the obtainment of17α-monoesters of cortexolone and of 9,11-dehydrocortexolone;furthermore, it also discloses the existence of several crystallineforms of cortexolone 17α-propionate, namely crystalline form I, form II,form III and hydrate form IV, and certain processes to obtain them.

Cyproterone acetate (abbreviated as CPA), is a synthetic steroid, whichwas considered as the standard therapy for the treatment ofandrogen-sensitive tumors, especially prostate cancer. The standardtherapy with Cyproterone Acetate resulted quite ineffective in thetumors with reduced, or absent, expression of Androgen Receptor (Br. J.Cancer (1989), 60, 789-792).

It is known in the art that the presence of 17α-esterification confersto cortexolone 17α-esters different antiandrogenic activities,demonstrated in animals (Celasco et al., Arzneim-Forsch 2005; 5: 581-7).

It has now been surprisingly found that cortexolone17α(alpha)-monoesters, 21-monoesters and 17α(alpha),21-diesters haveunexpected antitumor effects, both in isolated cancer cell lines invitro and in xenograft prostate and pancreatic cancer model in vivo intothe animals.

The antitumor effect of the invention was evident both in carcinomacells harboring Androgen Receptor (AR⁺), such as in the case of prostatecancer cells LNCaP or pancreatic cancer cells Panc1, and, verysurprisingly, also in cells with absent, or reduced, expression of theAndrogen Receptor (AR⁺), as prostate cancer cells PC3, or pancreaticcancer cells MiaPaca. The antitumor effect of the invention was alsoevident in mammary carcinomas (AR⁻), and gastrointestinal tractcarcinomas (AR⁻).

BRIEF DESCRIPTION OF THE FIGURES

The invention will now be described by the following non-limitingfigures and examples.

FIG. 1: Average fold change in pancreatic tumor volume, measuredrelative to the start of subcutaneous (SC) treatment, in the xenograftanimal model of nude mice (MiaPaca pancreatic cell line) withcortexolone 17α-benzoate (in the figure referred to as “06” and as“CB-03-06”) at low dose (230 μM) and at high dose (1150 μM). Referenceto “Vehicle” is a control treated group with 0.4% (v/v) tween 80 and0.5% (w/v) carboxymethylcellulose in normal saline. Mice were treatedwith the compound and vehicle SC daily for 28 consecutive days. The stoptreatment arrow refers to the day when the treatment was ended.

FIG. 2: Average fold change in pancreatic tumor volume, measuredrelatively to the start of SC treatment, in the xenograft animal modelof nude mice (MiaPaca pancreatic cell line) with cortexolone17α-valerate-21-propionate (in the figure referred to as “10” and as“CB-03-10”) at low dose (230 μM) and at high dose (1150 μM). Referenceto “Vehicle” is a control treated group with 0.4% (v/v) tween 80 and0.5% (w/v) carboxymethylcellulose in normal saline. Mice were treatedwith the compound and vehicle SC daily for 28 consecutive days. The stoptreatment arrow refers to the day when the treatment was ended.

FIG. 3: Average fold change in pancreatic tumor volume relative to thestart of SC treatment in the animal model of nude mice (MiaPacapancreatic cell line) treated with Cyproterone Acetate (in the figurereferred to as CPA), cortexolone 17α-valerate-21-propionate (in thefigure referred to as “10”) and cortexolone 17α-benzoate (in the figurereferred to as “06”) (each compound at low dose and at high dose) andwith vehicle (i.e. 0.4% (v/v) tween 80 and 0.5% (w/v)carboxymethylcellulose in normal saline) treated control group. All micewere treated with the compound and vehicle SC daily for 28 consecutivedays (days treatment). The stop treatment arrow refers to the day whenthe treatment was ended.

FIG. 4: Graph showing the P values Vs vehicle (i.e. 0.4% (v/v) tween 80and 0.5% (w/v) carboxymethylcellulose in normal saline) treated controlgroup of the best doses from FIG. 3. All mice were treated with thecompound and vehicle SC daily for 28 consecutive days (days treatment).The stop treatment arrow refers to the day when the treatment was ended.

FIGS. 5A and 5B: Dose Titration of cytotoxicity of cortexolone-derivedcompounds in human prostate (A) and pancreatic (B) cancer cell lines.

FIGS. 6A-6D: Androgen Receptor Expression levels on cancer cell lines:(A) PC-3 prostate; (B) LNCaP prostate; (C): MiaPaca-2 pancreatic; and(D): Panc-1 pancreatic.

FIG. 7: CB-03-10 and CB-03-05 glucocorticoid_antagonist activity.Mifepristone is used as positive control (potent glucocorticoidantagonist)

FIG. 8: CB-03-10 and CB-03-05 glucocorticoid agonist activity,Dexametasone (Dex) is used as positive control (potent glucocorticoidagonist)

FIG. 9: CB-03-10 induction of apoptosis in MiaPaca2 cells.

FIG. 10: Induction of cell cycle arrest by different concentrations ofCB-03-10 in MiaPaca2 cells.

FIGS. 11A-11C; Time course of caspases activation in MiaPaca2 Cells(8-24-48 hours). 20 μM (striped bars) or 50 μM (solid bars) indicate thecompound's concentrations. Gemcitabine is a potent anti pancreaticcytotoxic drug used and positive control. Caspases: (A): caspase 8; (B):caspase 9; and (C): caspase 3/4.

FIGS. 12A-12C: Caspase activation on LNCaP prostate cancer cell lines.Caspases: (A): caspase 8; (B): caspase 9; and (C): caspase 3/4.

FIGS. 13A and 13B: In vitro metabolism of CB-03-10 and CB-03-05 in (A)Human and (B) Rat plasma.

FIG. 14: CB-03-10 and CB-03-05 pharmacokinetics evaluated in vivo inplasma of mice after subcutaneous and oral administration.

FIG. 15: CB-03-10 in vivo anti-tumor activity on mouse xenograft modelof pancreatic cancer when administered subcutaneously.

FIG. 16: CB-03-10 in vivo anti-tumor activity in a mouse xenograft modelof prostate cancer when administered by oral gavage. Enzalutamide (Enza)a potent anti prostate cytotoxic drug is used as positive control. Ourresults show that the CB-03-10 Vs. vehicle is statisticallysignificantly different from day 7 up to day 60. In contrast,Enzalutamide Vs. vehicle is statistically significant only on days 14and 25. This comparison shows that CB-03-10 reaches a high statisticalsignificance versus vehicle whereas Enzalutamide does not reachstatistical significance versus vehicle.

FIG. 17; CB-03-10 and CB-03-05 inhibition of in vitro baseline PSAsecretion from LNCaP cancer cell lines.

FIG. 18: Androgen and Glucorticoid receptor expression in differentcancer cell lines.

DEFINITIONS

Unless otherwise defined, all terms of art, notations and otherscientific terminology used herein are intended to have the meaningscommonly understood by those skilled in the art to which this disclosurepertains. In some cases, terms with commonly understood meanings aredefined herein for clarity and/or for ready reference; thus, theinclusion of such definitions herein should not be construed torepresent a substantial difference over what is generally understood inthe art.

In particular, the terms “physiologically acceptable excipient” or“pharmaceutically acceptable excipient” herein refer to a substancedevoid of any pharmacological effect of its own and which does notproduce adverse reactions when administered to a mammal, preferably ahuman. Physiologically acceptable excipients are well known in the artand are disclosed, for instance in the Handbook of PharmaceuticalExcipients, sixth edition (2009), herein incorporated by reference.

The term “alkyl” as used herein means a saturated straight or branchedchain hydrocarbon.

The term “aryl” herein refers to aromatic mono- and poly-carbocyclicring systems, wherein the individual carbocyclic rings in thepoly-carbocyclic ring systems may be fused or attached to each other viaa single bond. Suitable “aryl” groups comprise, but are not limited to,phenyl, naphthyl, biphenyl, and the like.

The term “heteroaryl” herein refers to an aromatic mono- andpoly-carbocyclic ring system comprising at least a heteroatom in thering system, wherein said heteroatom is selected in the groupcomprising, but not limited to, nitrogen, sulphur, oxygen and the like,and wherein the individual cyclic rings in the poly-carbocyclic ringsystems may be fused or attached to each other via a single bond.Suitable “heteroaryl” groups comprise, but are not limited to, pyridyl,imidazolyl, pyrrolyl, furyl, benzimidazolyl, thiofuranyl and the like.

Aryl groups may be optionally substituted in at least one of the carbonatoms of the ring with a group selected from lower alkyl, lower alkenyl,lower haloalkyl, lower haloalkenyl, lower alkoxy, lower halcalkenyl,lower alkenyloxy, halogen, nitro, cyano, lower alkylthio, and the like.

Heteroaryl groups may be optionally substituted in at least one of thecarbon atoms or in at least one of the heteroatoms of the ring with agroup selected from lower alkyl, lower haloalkyl, lower alkoxy, loweralkenyl, lower halcalkenyl, lower alkenyloxy, halogen, nitro, cyano,lower alkylthio, and the like.

The term “approximately” herein refers to the range of the experimentalerror, which may occur in a measurement.

The terms “comprising”, “having”, “including” and “containing” are to beconstrued as open-ended terms (i.e. meaning “including, but not limitedto”) and are to be considered as including and/or providing support alsofor terms as “consist essentially of”, “consisting essentially of”,“consist of” or “consisting of”.

The terms “consist essentially of”, “consisting essentially of” are tobe construed as a semi-closed terms, meaning that no other ingredientswhich materially affects the basic and novel characteristics of theinvention are included (optional excipients may thus be included).

The terms “consists of”, “consisting of” are to be construed as a closedterm.

As used herein, the terms “therapeutically effective amount” and“effective amount” refer to an amount sufficient to elicit the desiredbiological response. In the present invention the desired biologicalresponse is to inhibit, reduce or ameliorate the severity, duration,progression, or onset of a disease, disorder or condition, prevent theadvancement, recurrence, or progression of a disease, disorder orcondition or a symptom associated with a disease, disorder or condition.

The precise amount of compound administered to a subject will depend onthe mode of administration, the type and severity of the disease,disorder or condition and on the characteristics of the subject, such asgeneral health, age, sex, body weight and tolerance to drugs. Theskilled artisan will be able to determine appropriate dosages dependingon these and other factors. Suitable dosages are known for approvedagents and can be adjusted by the skilled artisan according to thecondition of the subject, the type of condition(s) being treated and theamount of a compound described herein being used. In cases where noamount is expressly noted, an effective amount should be assumed. Forexample, compounds and pharmaceutical compositions described herein canbe administered to a subject in a dosage range from betweenapproximately 0.01 to 100 mg/kg body weight/day for therapeutictreatment.

As used herein, the terms “treat”, “treatment” and “treating” refer totherapeutic treatments includes the reduction or amelioration of theprogression, severity and/or duration of a disease, disorder orcondition, or the amelioration of one or more symptoms (specifically,one or more discernible symptoms) of a disease, disorder or condition,resulting from the administration of one or more therapies (e.g., one ormore therapeutic agents such as a compound or composition of theinvention). In specific embodiments, the therapeutic treatment includesthe amelioration of at least one measurable physical parameter of adisease, disorder or condition. In other embodiments the therapeutictreatment includes the inhibition of the progression of a condition,either physically by, e.g., stabilization of a discernible symptom,physiologically by, e.g., stabilization of a physical parameter, orboth. In other embodiments the therapeutic treatment includes thereduction or stabilization of a disease, disorder or condition. The term“curative treatment” as used herein refers to a treatment that aims tocure a disease or to improve symptoms associated with a disease.

The term “palliative treatment” as used herein refers to a treatment ortherapy that does not aim at curing a disease but rather at providingrelief.

The term “adjuvant treatment” as used herein refers to a treatment thatis given in addition to the primary, main or initial treatment.

The term “neoadjuvant treatment” as used herein refers to a treatmentthat is given before a main treatment, with the aim of reducing the sizeor extent of a tumor, thus reducing the consequences of a more extensivetreatment technique that would be required if the tumor wasn't reducedin size or extent.

As described herein, compounds of the invention may optionally besubstituted with one or more substituents, such as illustrated generallybelow, or as exemplified by particular species of the invention. It willbe appreciated that the phrase “optionally substituted” is usedinterchangeably with the phrase “substituted or unsubstituted.” Ingeneral, the term “substituted”, whether preceded by the term“optionally” or not, refers to the replacement of one or more hydrogenradicals in a given structure with the radical of a specifiedsubstituent. Unless otherwise indicated, an optionally substituted groupmay have a substituent at each substitutable position of the group. Whenmore than one position in a given structure can be substituted with morethan one substituent selected from a specified group, the substituentmay be either the same or different at each position. When the term“optionally substituted” precedes a list, said term refers to all of thesubsequent substitutable groups in that list. If a substituent radicalor structure is not identified or defined as “optionally substituted”,the substituent radical or structure is unsubstituted.

Selection of substituents and combinations of substituents envisioned bythis invention are those that result in the formation of stable orchemically feasible compounds. The term “stable”, as used herein, refersto compounds that are not substantially altered when subjected toconditions to allow for their production, detection, and, specifically,their recovery, purification, and use for one or more of the purposesdisclosed herein. In some embodiments, a stable compound or chemicallyfeasible compound is one that is not substantially altered when kept ata temperature of 40° C. or less, in the absence of moisture or otherchemically reactive conditions, for at least a week. Only those choicesand combinations of substituents that result in a stable structure arecontemplated. Such choices and combinations will be apparent to those ofordinary skill in the art and may be determined without undueexperimentation.

The term “simultaneous, separate or sequential administration” hereinrefers to administration of the first and second compound at the sametime or in such a manner that the two compounds act in the patient'sbody at the same time or administration of one compound after the othercompound in such a manner to provide a therapeutic effect. In someembodiments the compounds are taken with a meal. In other embodiments,the compounds are taken after a meal, such as 30 minutes or 60 minutesafter a meal. In some embodiments, one compound is administered to apatient for a time period followed by administration of the othercompound.

As used herein, the terms “subject” and “patient” are usedinterchangeably. The terms “subject” and “patient” refer to an animal(e.g., a bird such as a chicken, quail or turkey, or a mammal),specifically a “mammal” including a non-primate (e.g., a cow, pig,horse, sheep, rabbit, guinea pig, rat, cat, dog, and mouse) and aprimate (e.g., a monkey, chimpanzee and a human), and more specificallya human. In one embodiment, the subject is a human.

DETAILED DESCRIPTION OF THE INVENTION

Now it has been surprisingly discovered that some cortexolonederivatives have therapeutically interesting anti-tumoral properties,against tumors, preferably epithelial and/or hormone-dependent tumors.

According to the general concept, the invention is represented by thecompounds of formula (I)

wherein R is hydrogen or C(O)—R₁, wherein R₁ is a linear alkyl chaincontaining 2 to 5 carbon atoms, and wherein R′ is a linear alkyl chaincontaining 3 to 6 carbon atoms or an optionally substituted aryl groupor an optionally substituted heteroaryl group.

Preferred compounds of formula (I) are those wherein R is hydrogen orC(O)—R₁, wherein R₁ CH₂CH₃ and wherein R′ is —(CH₂)₃—CH₃ or phenyl.

An object of the present invention is represented by the compounds offormula I

wherein R is C(O)—R₁, wherein R₁ is hydrogen or a linear alkyl chaincontaining 2 to 5 carbon atoms, and wherein R′ is a linear alkyl chaincontaining 3 to 6 carbon atoms or an optionally substituted aryl groupor an optionally substituted heteroaryl group wherein R₁ and R′ are notthe same.

An object of the present invention is represented by the compounds offormula I

wherein R is C(O)—R₁, wherein R₁ is a linear alkyl chain containing 2 to5 carbon atoms, and wherein R′ is a linear alkyl chain containing 3 to 6carbon atoms or an optionally substituted aryl group or an optionallysubstituted heteroaryl group wherein R₁ and R′ are not the same.Preferred compounds of formula (I) are those wherein R is C(O)—R₁,wherein R₁ is hydrogen or CH₂CH₃ and wherein R′ is —(CH₂)₃—CH₃ orphenyl, wherein R₁ and R′ are not the same.

Preferred compounds of formula (I) are those wherein R is C(O)—R₁,wherein R₁ is CH₂CH₃ and wherein R′ is —(CH₂)₃—CH₃ or phenyl, wherein R₁and R′ are not the same. The most preferred compound of formula (I) isthe compound wherein R is C(O)—R₁, wherein R₁ is CH₂CH₃ and wherein R′is —(CH₂)₃—CH₃, that is cortexolone 17α-valerate-21-propionate (hereinalso referred to as “10” or as “CB-03-10”), whose formula is reportedherein below:

Unless otherwise indicated, structures depicted herein are also meant toinclude all isomeric (e.g., enantiomeric, diastereomeric, cis-trans,conformational, and rotational) forms of the structure. For example, theR and S configurations for each asymmetric center, (Z) and (E) doublebond isomers, and (Z) and (E) conformational isomers are included inthis invention, unless only one of the isomers is drawn specifically. Aswould be understood to one skilled in the art, a substituent can freelyrotate around any rotatable bonds. Therefore, single stereochemicalisomers as well as enantiomeric, diastereomeric, cis/trans,conformational, and rotational mixtures of the present compounds arewithin the scope of the invention.

Unless otherwise indicated, all tautomeric forms of the compounds of theinvention are within the scope of the invention.

Additionally, unless otherwise indicated, structures depicted herein arealso meant to include compounds that differ only in the presence of oneor more isotopically enriched atoms. For example, compounds having thepresent structures except for the replacement of hydrogen by deuteriumor tritium, or the replacement of a carbon by a ¹³C- or ¹⁴C-enrichedcarbon are within the scope of this invention. Such compounds areuseful, for example, as analytical tools or probes in biological assays.Such compounds, especially deuterium analogues, can also betherapeutically useful.

The compounds of the invention are defined herein by their chemicalstructures and/or chemical names Where a compound is referred to by botha chemical structure and a chemical name, and the chemical structure andchemical name conflict, the chemical structure is determinative of thecompound's identity.

Pharmaceutically Acceptable Salts, Solvates, Clatrates, Prodrugs andOther Derivatives

The compounds described herein can exist in free form, or, whereappropriate, as salts. Those salts that are pharmaceutically acceptableare of particular interest since they are useful in administering thecompounds described below for medical purposes. Salts that are notpharmaceutically acceptable are useful in manufacturing processes, forisolation and purification purposes, and in some instances, for use inseparating stereoisomeric forms of the compounds of the invention orintermediates thereof.

As used herein, the term “pharmaceutically acceptable salt” refers tosalts of a compound which are, within the scope of sound medicaljudgment, suitable for use in contact with the tissues of humans andlower animals without undue side effects, such as, toxicity, irritation,allergic response and the like, and are commensurate with a reasonablebenefit/risk ratio.

Pharmaceutically acceptable salts are well known in the art. Forexample, S. M. Berge et al., describe pharmaceutically acceptable saltsin detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, incorporatedherein by reference. Pharmaceutically acceptable salts of the compoundsdescribed herein include those derived from suitable inorganic andorganic acids and bases. These salts can be prepared in situ during thefinal isolation and purification of the compounds.

It should be understood that this invention includesmixtures/combinations of different pharmaceutically acceptable salts andalso mixtures/combinations of compounds in free form andpharmaceutically acceptable salts.

In addition to the compounds described herein, pharmaceuticallyacceptable solvates (e.g., hydrates) and clathrates of these compoundsmay also be employed in compositions to treat or prevent the hereinidentified disorders.

As used herein, the term “pharmaceutically acceptable solvate,” is asolvate formed from the association of one or more pharmaceuticallyacceptable solvent molecules to one of the compounds described herein.The term solvate includes hydrates (e.g., hemihydrate, monohydrate,dihydrate, trihydrate, tetrahydrate, and the like).

As used herein, the term “hydrate” means a compound described herein ora salt thereof that further includes a stoichiometric ornon-stoichiometric amount of water bound by non-covalent intermolecularforces.

As used herein, the term “clathrate” means a compound described hereinor a salt thereof in the form of a crystal lattice that contains spaces(e.g., channels) that have a guest molecule (e.g., a solvent or water)trapped within.

In addition to the compounds described herein, pharmaceuticallyacceptable derivatives or prodrugs of these compounds may also beemployed in compositions to treat or prevent the herein identifieddisorders.

A “pharmaceutically acceptable derivative or prodrug” includes anypharmaceutically acceptable ester, salt of an ester or other derivativeor salt thereof of a compound described herein which, uponadministration to a recipient, is capable of providing, either directlyor indirectly, a compound described herein or an inhibitory activemetabolite or residue thereof. Particularly favored derivatives orprodrugs are those that increase the bioavailability of the compoundswhen such compounds are administered to a patient (e.g., by allowing anorally administered compound to be more readily absorbed into the blood)or which enhance delivery of the parent compound to a biologicalcompartment (e.g., the brain or lymphatic system) relative to the parentspecies.

Pharmaceutically acceptable prodrugs of the compounds described hereininclude, without limitation, esters, amino acid esters, phosphateesters, metal salts and sulfonate esters.

Medical Uses

Another object of the present invention is represented by the compoundsof formula (I) for use as a medicament.

Another object of the present invention is cortexolone 17α-valerate(herein also referred to as “05” or as “CB-03-05”), represented by:

for use as a medicament.

In one embodiment the compounds of the invention for use as a medicamentare Cortexolone 17α-valerate-21-propionate (CB-03-10) and Cortexolone17α-Valerate (CB-03-05).

In another embodiment the present invention is compositions andpharmaceutical compositions comprising compounds of formula (I) for useas a medicament.

In one embodiment the present invention is compositions andpharmaceutical compositions comprising compounds Cortexolone17α-valerate-21-propionate (CB-03-10) and Cortexolone 17α-Valerate(CB-03-05) for use as a medicament.

In another aspect, the invention relates to said compounds of formula(I), including Cortexolone 17α-valerate-21-propionate (CB-03-10) andCortexolone 17α-Valerate (CB-03-05) for use as a Glucocorticoid Receptor(GR) modulator, preferably a glucocorticoid antagonist.

In another embodiment the present invention is compositions andpharmaceutical compositions comprising compounds of formula (I) for useas a Glucocorticoid Receptor (GR) modulator, preferably a glucocorticoidantagonist.

In another embodiment the present invention is compositions andpharmaceutical compositions comprising Cortexolone17α-valerate-21-propionate (CB-03-10) and Cortexolone 17α-Valerate(CB-03-05) for use as a Glucocorticoid Receptor (GR) modulator,preferably a glucocorticoid antagonist.

In yet another aspect, the invention relates to said compounds offormula (I) for use in treating a disease or disorder mediated byglucocorticoid. In one embodiment the compounds of the invention intreating a disease or disorder mediated by glucocorticoid areCortexolone 17α-valerate-21-propionate (CB-03-10) and Cortexolone17α-Valerate (CB-03-05).

In still another aspect, the invention relates to said compounds offormula (I), including Cortexolone 17α-valerate-21-propionate (CB-03-10)and Cortexolone 17α-Valerate (CB-03-05) for use in the treatment ofprecancerous lesions, dysplasias, metaplasias and tumor diseases,including malignant neoplasias and metastasis; according to anotheraspect, such a treatment may be curative, adjuvant, neoadjuvant orpalliative.

Ideally, the compounds of formula (I), including Cortexolone17α-valerate-21-propionate (CB-03-10) and Cortexolone 17α-Valerate(CB-03-05) are for use as an anti-tumor agent.

In an embodiment, said tumor diseases are solid tumors, preferablyepithelial tumors, such as, by way of example, prostate carcinoma,mammary carcinoma, pancreatic carcinoma, lung carcinoma,gastrointestinal tract carcinoma (preferably colon carcinoma), kidneycancer, thyroid carcinoma, uterine carcinoma and adrenal carcinoma andthe like. In a preferred embodiment of the invention herein disclosed,said epithelial tumors are prostate carcinoma, pancreatic carcinoma(preferably exocrine pancreatic carcinoma), gastrointestinal tractcarcinoma (preferably colon carcinoma) and mammary carcinoma (preferablytriple negative breast cancer).

In a preferred embodiment of the invention herein disclosed, the tumordiseases are prostate cancer. In a preferred embodiment of the inventionherein disclosed, the prostate cancer is an adenocarcinoma. In apreferred embodiment of the invention herein disclosed, the tumordiseases are prostate cancer with absent or reduced expression of AR. Inanother preferred embodiment of the invention, the tumor diseases areprostate cancer with mutated or truncated Androgen Receptors.

Ideally, the compounds of formula (I), including Cortexolone17α-valerate-21-propionate (CB-03-10) and Cortexolone 17α-Valerate(CB-03-05) are for use as an anti-tumor agent where the tumor diseasesare prostate cancer with mutated or truncated Androgen Receptors. Oneparticularly advantageous use of the compounds of formula (I), includingCortexolone 17α-valerate-21-propionate (CB-03-10) and Cortexolone17α-Valerate (CB-03-05), is for use in the treatment of prostate cancersthat are or have become resistant to anti-androgen treatment, such asenzalutamide. This is a particularly advantageous embodiment of theinvention as it has recently been found that after 6 months of treatment30% of prostate cancers became resistant to enzalutamide because the ARhas mutated or changed. Interestingly these resistant cancer cellsupregulate the GR. The compounds of formula (I), including Cortexolone17α-valerate-21-propionate (CB-03-10) and Cortexolone 17α-Valerate(CB-03-05), can be used to treat such cancers as the activity is alsomediated through the GR.

In a preferred embodiment of the invention herein disclosed, theexocrine pancreatic carcinoma is an adenocarcinoma. In a preferredembodiment the exocrine pancreatic cancer with absent or reducedexpression of the AR.

In a preferred embodiment of the invention herein disclosed, saidepithelial tumor is gastrointestinal tract carcinoma (preferably coloncarcinoma).

In a still preferred embodiment of the invention herein disclosed, saidepithelial tumor is mammary carcinoma (preferably triple negative breastcancer). Optionally, the subject or patient being treated is anon-responder or a relapse to conventional therapy.

In a preferred embodiment, the present invention provides the compoundsof formula (I), wherein R is C(O)—R₁, wherein R₁ is hydrogen or CH₂CH₃and R′ is —(CH₂)₃—CH₃ or a phenyl group, wherein R₁ and R′ are not thesame, for use in the treatment of precancerous lesions, dysplasias,metaplasias and tumor diseases, including malignant neoplasias andmetastasis; according to another aspect, such a treatment may becurative, adjuvant, neoadjuvant or palliative. Ideally, the compound isfor use as an anti-tumor agent.

In a preferred embodiment, the present invention provides the compoundsof formula (I), wherein R is C(O)—R₁, wherein R₁ is CH₂CH₃ and R′ is—(CH₂)₃—CH₃ or a phenyl group, wherein R₁ and R′ are not the same, foruse in the treatment of precancerous lesions, dysplasias, metaplasiasand tumor diseases, including malignant neoplasias and metastasis;according to another aspect, such a treatment may be curative, adjuvant,neoadjuvant or palliative. Ideally, the compound is for use as ananti-tumor agent.

In another embodiment, the present invention provides the compound offormula (I) wherein R is C(O)—R₁, wherein R₁ CH₂CH₃ and wherein R′ is—(CH₂)₃—CH₃, that is cortexolone 17α-valerate-21-propionate (herein alsoreferred to as “10” or as “CB-03-10”), for use in the treatment ofprecancerous lesions, dysplasias, metaplasias and tumor diseases,including malignant neoplasias and metastasis; according to anotheraspect, such a treatment may be curative, adjuvant, neoadjuvant orpalliative. Ideally, the compound is for use as an anti-tumor agent.

In an embodiment, said tumor diseases are solid tumors, preferablyepithelial tumors, such as, by way of example, prostate carcinoma,mammary carcinoma, pancreatic carcinoma, lung carcinoma,gastrointestinal tract carcinoma (preferably colon carcinoma), kidneycancer, thyroid carcinoma, uterine carcinoma and adrenal carcinoma andthe like.

In a preferred embodiment of the invention herein disclosed, saidepithelial tumors are prostate carcinoma, pancreatic carcinoma(preferably exocrine pancreatic carcinoma), gastrointestinal tractcarcinoma (preferably colon carcinoma) and mammary carcinoma (preferablytriple negative breast cancer).

In a preferred embodiment of the invention herein disclosed, the tumordiseases are prostate cancer. In a preferred embodiment of the inventionherein disclosed, the prostate cancer is an adenocarcinoma. In apreferred embodiment of the invention herein disclosed, the tumordiseases are prostate cancer with mutated, absent or reduced expressionof the AR.

In a preferred embodiment of the invention herein disclosed, theexocrine pancreatic carcinoma is an adenocarcinoma. In a preferredembodiment the exocrine pancreatic cancer with absent or reducedexpression of the AR.

Another object of the present invention is represented by the compoundsof formula (I) for use in the manufacture of medicament for thetreatment of precancerous lesions, dysplasias, metaplasias and tumordiseases, including malignant neoplasias and metastasis; according toanother aspect, such a treatment may be curative, adjuvant, neoadjuvantor palliative. In one embodiment the compounds of the invention are foruse in the manufacture of a medicament for the treatment of precancerouslesions, dysplasias, metaplasias and tumor diseases, including malignantneoplasias and metastasis are Cortexolone 17α-valerate-21-propionate(CB-03-10) and Cortexolone 17α-Valerate (CB-03-05). Ideally, thecompounds of formula (I) are for use in the manufacture of an anti-tumoragent. In one embodiment the compounds of the invention for use in themanufacture of anti tumor agents are Cortexolone17α-valerate-21-propionate (CB-03-10) and Cortexolone 17α-Valerate(CB-03-05).

In an embodiment, said tumor diseases are solid tumors, preferablyepithelial tumors, such as, by way of example, prostate carcinoma,mammary carcinoma, pancreatic carcinoma, lung carcinoma,gastrointestinal tract carcinoma (preferably colon carcinoma), kidneycancer, thyroid carcinoma, uterine carcinoma and adrenal carcinoma andthe like.

In another aspect, the invention relates to said compounds of formula(I) for use in the manufacture of a medicament for treating a disease ordisorder mediated by glucocorticoid.

In one aspect, the invention herein disclosed provides a method fortreating precancerous lesions, dysplasias, metaplasias and tumordiseases, including malignant neoplasias and metastasis, said methodcomprising the administration of an effective amount of a compound offormula (I):

wherein R is C(O)—R₁, wherein R₁ is hydrogen or a linear alkyl chaincontaining 2 to 5 carbon atoms, and wherein R′ is a linear alkyl chaincontaining 3 to 6 carbon atoms or an optionally substituted aryl groupor an optionally substituted heteroaryl group, wherein R₁ and R′ are notthe same, to a subject in need thereof. Preferably, said subject is amammal. Preferably, said mammal is a human.

In one aspect, the invention herein disclosed provides a method fortreating precancerous lesions, dysplasias, metaplasias and tumordiseases, including malignant neoplasias and metastasis, said methodcomprising the administration of an effective amount of a compound offormula (I):

wherein R is C(O)—R₁, wherein R₁ is a linear alkyl chain containing 2 to5 carbon atoms, and wherein R′ is a linear alkyl chain containing 3 to 6carbon atoms or an optionally substituted aryl group or an optionallysubstituted heteroaryl group, wherein R₁ and R′ are not the same, to asubject in need thereof. Preferably, said subject is a mammal.Preferably, said mammal is a human.

In an embodiment, said tumor diseases are solid tumors, particularlyepithelial tumors, such as, by way of example, prostate carcinoma,mammary carcinoma, uterine carcinoma, pancreatic carcinoma, lungcarcinoma, gastro-intestinal tract carcinoma (preferably coloncarcinoma), kidney cancer, thyroid carcinoma, and adrenal carcinoma andthe like.

In an embodiment, said tumor diseases are solid tumors, particularlyepithelial tumors, such as, by way of example, prostate carcinoma,mammary carcinoma, uterine carcinoma, pancreatic carcinoma, lungcarcinoma, gastro-intestinal tract carcinoma (preferably coloncarcinoma), kidney cancer, thyroid carcinoma, uterine carcinoma andadrenal carcinoma and the like.

In a preferred embodiment of the invention herein disclosed, saidepithelial tumors are prostate carcinoma or pancreatic carcinoma, morepreferably exocrine pancreatic carcinoma, or mammary carcinoma, such as,triple negative breast cancer. In a preferred embodiment of theinvention herein disclosed, the tumor diseases are prostate cancer withabsent or reduced expression of AR. In another preferred embodiment ofthe invention, the tumor diseases are prostate cancer with mutated ortruncated Androgen Receptors. In this manner, the prostate cancer thatmay be treated according to the invention may be or have becomeresistant to anti-androgen targeted therapy, such as enzalutamide.

According to a preferred embodiment, said method comprises theadministration of an effective amount of a compound of formula (I)wherein R is C(O)—R₁, wherein R₁ is hydrogen or CH₂CH₃ and R′ is—(CH₂)₃—CH₃ or a phenyl group wherein R₁ and R′ are not the same, to amammal in need thereof.

According to a preferred embodiment, said method comprises theadministration of an effective amount of a compound of formula (I)wherein R is C(O)—R₁, wherein R₁ is CH₂CH₃ and R′ is —(CH₂)₃—CH₃ or aphenyl group wherein R₁ and R′ are not the same, to a mammal in needthereof. In a most preferred embodiment, said method comprises theadministration of an effective amount of a compound of formula (I)wherein R is C(O)—R₁, R₁ is CH₂CH₃ and R′ is (CH₂)₃—CH₃, that iscortexolone 17α-valerate-21-propionate (CB-03-10).

In one aspect, the invention herein disclosed provides a method fortreating precancerous lesions, dysplasias, metaplasias and tumordiseases, including malignant neoplasias and metastasis, said methodcomprising the administration of an effective amount of cortexolone17α-valerate (CB-03-05), to a subject in need thereof. Preferably, saidsubject is a mammal. Preferably, said mammal is a human.

In an embodiment, said tumor diseases are solid tumors, particularlyepithelial tumors, such as, by way of example, prostate carcinoma,mammary carcinoma, uterine carcinoma, pancreatic carcinoma, lungcarcinoma, gastro-intestinal tract carcinoma (preferably coloncarcinoma), kidney cancer, thyroid carcinoma, and adrenal carcinoma andthe like.

In a preferred embodiment of the invention herein disclosed, saidepithelial tumors are prostate carcinoma or pancreatic carcinoma, morepreferably exocrine pancreatic carcinoma, or mammary carcinoma, such as,triple negative breast cancer. The compounds of the present inventioncan be used in different therapeutic applications, especially oncologicapplications. More in details, the compounds according to the inventionherein disclosed have been found particularly effective for the curativeor adjuvant, or neoadjuvant or palliative treatment of pancreaticcarcinoma, preferably exocrine pancreatic carcinoma, and prostaticcarcinoma. An illustration of the pharmacological properties of thecompounds of the invention will be found hereafter in the experimentalsection.

The compounds of formula (I) may be prepared according to anyconventional method, for instance by the processes disclosed inWO03/014141 and in WO2009/019138, the contents of each of which areherein incorporated by reference their entireties. According to anembodiment of the invention, cortexolone 17α-valerate-21-propionate(CB-03-10) and cortexolone 17α-valerate (CB-03-05) can be preparedaccording to the method disclosed in example 11.

Pharmaceutical Compositions

The compounds described herein can be formulated into pharmaceuticalcompositions that further comprise a pharmaceutically acceptablecarrier, diluent, adjuvant or vehicle. In one embodiment, the presentinvention relates to a pharmaceutical composition comprising a compoundof the invention described herein, and a pharmaceutically acceptablecarrier, diluent, adjuvant or vehicle. In one embodiment, the presentinvention is a pharmaceutical composition comprising an effective amountof a compound of the present invention or a pharmaceutically acceptablesalt thereof and a pharmaceutically acceptable carrier, diluent,adjuvant or vehicle. Pharmaceutically acceptable carriers include, forexample, pharmaceutical diluents, excipients or carriers suitablyselected with respect to the intended form of administration, andconsistent with conventional pharmaceutical practices.

Another object of the present invention is represented by apharmaceutical composition comprising, as active ingredient, at leastone compound of formula (I), wherein R is —C(O)—R₁, wherein R₁ ishydrogen or a linear alkyl chain containing 2 to 5 carbon atoms, andwherein R′ is a linear alkyl chain containing 3 to 6 carbon atoms or anoptionally substituted aryl group or an optionally substitutedheteroaryl group, wherein R₁ and R′ are not the same in association withat least one physiologically acceptable excipient.

Another object of the present invention is represented by apharmaceutical composition comprising, as active ingredient, at leastone compound of formula (I), wherein R is C(O)—R₁, wherein R₁ is alinear alkyl chain containing 2 to 5 carbon atoms, and wherein R′ is alinear alkyl chain containing 3 to 6 carbon atoms or an optionallysubstituted aryl group or an optionally substituted heteroaryl group,wherein R₁ and R′ are not the same in association with at least onephysiologically acceptable excipient. According to a preferredembodiment of the invention said pharmaceutical composition comprises,as active ingredient, at least one compound of formula (I) wherein R isC(O)—R₁, wherein R₁ is hydrogen or —CH₂CH₃ and R′ is —(CH₂)₃—CH₃ or aphenyl group, wherein R₁ and R′ are not the same.

According to a preferred embodiment of the invention said pharmaceuticalcomposition comprises, as active ingredient, at least one compound offormula (I) wherein R is C(O)—R₁, wherein R₁ is CH₂CH₃ and R′ is—(CH₂)₃—CH₃ or a phenyl group, wherein R₁ and R′ are not the same.According to a most preferred embodiment, said pharmaceuticalcomposition comprises, as active ingredient, cortexolone17α-valerate-21-propionate (CB-03-10), in association with at least onephysiologically acceptable excipient. Another object of the presentinvention is represented by a pharmaceutical composition comprising, asactive ingredient, cortexolone 17α-valerate (CB-03-05) in associationwith at least one physiologically acceptable excipient.

In a further object, the compounds and pharmaceutical compositions ofthe invention are for use as a medicament, preferably in the treatmentof precancerous lesions, dysplasias, metaplasias and tumor diseases,including malignant neoplasias and metastasis; according to anotheraspect, such a treatment may be curative, adjuvant, neoadjuvant orpalliative. In this manner, they are used as anti-tumor agents.Preferably, said tumor diseases are solid tumors. More preferably, saidsolid tumors are epithelial tumors, such as, by way of example, prostatecarcinoma, mammary carcinoma, pancreatic carcinoma, lung carcinoma,gastro-intestinal tract carcinoma (preferably colon carcinoma), kidneycancer, thyroid carcinoma, uterine carcinoma and adrenal carcinoma andthe like. In a preferred embodiment of the invention herein disclosed,said epithelial tumors are prostate carcinoma and pancreatic carcinoma,more preferably exocrine pancreatic carcinoma or mammary carcinoma, suchas, triple negative breast cancer. In another preferred embodiment ofthe invention, the tumor diseases are prostate cancer with mutated ortruncated Androgen Receptors. Ideally, the pharmaceutical compositionscomprising compounds of formula (I), including Cortexolone17α-valerate-21-propionate (CB-03-10) and Cortexolone 17α-Valerate(CB-03-05), for use as an anti-tumor agent where the tumor diseases areprostate cancer with mutated or truncated Androgen Receptors. In thismanner, the prostate cancer that may be treated according to theinvention may be or have become resistant to anti-androgen targetedtherapy, such as enzalutamide.

As described above, in another embodiment, there is providedpharmaceutical compositions comprising compounds of formula (I),including Cortexolone 17α-valerate-21-propionate (CB-03-10) andCortexolone 17α-Valerate (CB-03-05), for use as a GlucocorticoidReceptor (GR) modulator, preferably a glucocorticoid antagonist.

The pharmaceutical compositions of the invention can be in solid form,such as, by way of example, powders, freeze-dried powders, granules,pellets, tablets or capsules. If desired, certain sweetening, flavoringor coloring agents may also be added. The compounds of the invention canalso be in microencapsulated form with one or more excipients. The soliddosage forms of tablets, capsules, pills, and granules can be preparedwith coatings and shells such as enteric coatings and other coatingswell known in the pharmaceutical formulating art. Appropriate excipientsfor solid pharmaceutical compositions can be selected, without anylimitation, among the categories known to a person skilled in the artsuch as adsorbents, fillers, surfactants, compression aids, binders,lubricants, disintegrants, diluents, disgregants, flow promoting agents,freeze-drying agents, glidants, lyophilization aids, film-formingagents, dyes, antioxidants, and the like. By way of example, suitableexcipients for solid pharmaceutical compositions can be selected, in anon-limiting way, from calcium phosphate, magnesium stearate, talc,sugars, lactose, dextrin, starch, gelatin, cellulose and derivativesthereof, polyvinylpyrrolidone, coating agents, dyes and wax. Any mixtureof these excipients can be properly used according to the invention.

According to the invention, solid pharmaceutical compositions such astablets, granules, pellets, capsules and the like, can be formulated asimmediate release forms or as delayed release forms or as controlledrelease forms or as extended release forms or as prolonged releaseforms, and are suitable for administration by the oral, or sublingualadministration route or as an implant.

The controlled, extended and/or prolonged composition may be preparedaccording to to any conventional method or system, for instanceaccording to WO00/76478 herein incorporated by reference in it'sentirety.

The pharmaceutical compositions of the invention can also be in liquidform, for example, solutions, emulsions, suspensions or syrups. Liquiddosage forms for oral administration include, but are not limited to,pharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. In addition to the active compounds,the liquid dosage forms may contain inert diluents commonly used in theart. Besides inert diluents, the oral compositions can also includeadjuvants such as wetting agents, emulsifying and suspending agents,sweetening, flavoring, and perfuming agents. Appropriate excipients forliquid pharmaceutical composition can be selected, without anylimitation, among the categories well known to a person skilled in theart, such as solvents, co-solvents, oleaginous vehicles, bufferingagents, surfactants, emulsifying agents, solubility enhancing agents,suspending agents, solubilizing agents, chelating agents, acidifyingagents, alkalinizing agents, antioxidants, preservatives, osmoticagents, tonicity agents, viscosity controlling agents and the like. Byway of example, suitable pharmaceutical excipients for liquidpreparation can be selected from water for injections, organic solventsor co-solvents such as ethanol, glycols and glycerol and mixturesthereof, natural oils such as soybean oil, medium-chain triglycerides,polyoxyl 15-hydroxystearate, polysorbate 80, polyoxyl 35-castor oil,sodium chloride, sodium phosphate, potassium phosphate, and the like.According to the invention, said liquid pharmaceutical compositions canbe sterile or non-sterile. In one embodiment, the liquid pharmaceuticalcompositions are terminally sterilized by means of a technique wellknown to a person skilled in the art, such as dry heat sterilization,moist heat sterilization, gamma radiation, e-beam sterilization and thelike. In another embodiment, the liquid pharmaceutical compositions aresterilized by sterile filtration and aseptically filled in the finalprimary packaging containers. The liquid pharmaceutical compositionsaccording to the invention herein disclosed can be used for injections,infusions or perfusions such as intravenous, intramuscular,intraperitoneal, subcutaneous or intratumoral administration.

Administration Methods

The compounds and compositions described herein may be administeredorally, parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir. The term “parenteral”as used herein includes, but is not limited to, subcutaneous,intravenous, intramuscular, intra-articular, intra-synovial,intrasternal, intrathecal, intrahepatic, intralesional and intracranialinjection or infusion techniques.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent. The injectable formulations can be sterilized, forexample, by filtration through a bacterial-retaining filter, or byincorporating sterilizing agents in the form of sterile solidcompositions which can be dissolved or dispersed in sterile water orother sterile injectable medium prior to use.

Sterile injectable forms of the compounds and compositions describedherein may be aqueous or oleaginous suspension. These suspensions may beformulated according to techniques known in the art using suitabledispersing or wetting agents and suspending agents. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a nontoxic parenterally-acceptable diluent or solvent.

The compounds for use in the methods of the invention can be formulatedin unit dosage form. The term “unit dosage form” refers to physicallydiscrete units suitable as unitary dosage for subjects undergoingtreatment, with each unit containing a predetermined quantity of activematerial calculated to produce the desired therapeutic effect,optionally in association with a suitable pharmaceutical carrier. Theunit dosage form can be for a single daily dose or one of multiple dailydoses (e.g., about 1 to 4 or more times per day). When multiple dailydoses are used, the unit dosage form can be the same or different foreach dose.

According to the invention, the compounds of formula (I) or thepharmaceutical compositions comprising the said compounds are preferablyadministered by intravenous injection, more preferably through aninfusion bag or a syringe or a pump catheter, or by intramuscularinjection, or by subcutaneous injection, or per os (by mouth) in formsof tablets or capsules.

According to an embodiment, said pharmaceutical composition is in liquidform and is suitable for injection, and comprise a cortexolone-derivedcompound of formula (I) in an amount ranging from 0.1% to 50.0% weightto volume (w/v), preferably from 0.25% to 25% w/v, more preferably from0.5% to 10% w/v, much more preferably from 1% to 5% w/v.

According to another embodiment, said pharmaceutical composition is insolid form and comprises a cortexolone-derived compound of formula (I)in an amount ranging from 0.1% to 50% weight to weight (w/w), preferablyfrom 0.5% to 40% w/w, more preferably from 1% to 30% w/w.

The amount of the at least one compound of formula (I) in saidpharmaceutical composition is such that an effective dosage level can beobtained upon administration to a mammal suffering of precancerouslesions, dysplasias, metaplasias and tumor diseases, including malignantneoplasias and metastasis. The compounds of formula (I) and thepharmaceutical composition comprising the same as antitumor activeingredients for use in the curative or adjuvant, or neoadjuvant orpalliative treatment of precancerous lesions, dysplasias, metaplasiasand tumor diseases, including malignant neoplasias and metastasis, arepreferably administered to a mammal, said mammal being a human or ananimal, preferably a human.

Combination Therapy

According to another embodiment, the compounds, compositions andpharmaceutical compositions may contain at least another activeingredient, preferably a chemotherapeutic active ingredient, as acombination for simultaneous, separate or sequential administration.

In certain embodiments, the compounds of formula (I) and thepharmaceutical composition comprising at least one compound of formula(I) and at least one physiologically acceptable excipient according tothe invention can be used in combination therapy with at least one otherdrug, especially a chemotherapeutic drug.

In certain embodiments, the compounds of the invention can beadministered concurrently with the administration of another drug,especially a chemotherapeutic drug. In certain embodiments, thecompounds of the invention can be administered prior to or subsequent toadministration of another drug, especially a chemotherapeutic drug. Saidat least one other drug, especially a chemotherapeutic drug, can beeffective for treating the same or different disease, disorder, orcondition.

Methods of the present invention include administration of one or morecompounds of formula (I) or pharmaceutical compositions comprising atleast a compound of formula (I) of the present invention and at leastanother drug, preferably a chemotherapeutic drug, provided that thecombined administration does not inhibit the therapeutic efficacy of theone or more compounds of the present invention and/or does not producenon-acceptable adverse combination effects.

Cortexolone 17α-Valerate (Herein Also Referred to as “05” or as“CB-03-05”)

As described above, another object of the present invention iscortexolone 17α-valerate (herein also referred to as “05” or as“CB-03-05”), represented by:

for use as a medicament.

Ideally, cortexolone 17α-valerate is for use in the treatment ofprecancerous lesions, dysplasias, metaplasias and tumor diseases,optionally including malignant neoplasias and metastasis. Preferably,cortexolone 17α-valerate is for use as an anti-tumor agent.

Preferably, the tumor diseases are solid tumors, preferably epithelialtumors. The epithelial tumors may be selected from prostate carcinoma;mammary carcinoma; pancreatic carcinoma (preferably exocrine pancreaticcancer); lung carcinoma; gastrointestinal tract carcinoma, such as coloncarcinoma; kidney cancer; thyroid carcinoma; uterine carcinoma; andadrenal carcinoma.

According to one embodiment, the epithelial tumor is a prostatecarcinoma. In another preferred embodiment of the invention, the tumordiseases are prostate cancer with mutated or truncated AndrogenReceptors. In this manner, the prostate cancer that may be treatedaccording to the invention may be or have become resistant toanti-androgen targeted therapy, such as enzalutamide.

According to another embodiment, the epithelial tumors are pancreaticcarcinoma, preferably exocrine pancreatic carcinoma.

According to one embodiment, the epithelial tumors are mammarycarcinoma, preferably triple negative breast cancer (TNBC). In oneembodiment, the mammary carcinoma is triple negative breast cancer andthe subject is a relapsed or a non-responder to conventional therapy.

According to another embodiment, the epithelial tumors aregastrointestinal tract carcinoma, such as colon carcinoma.

According to another embodiment, cortexolone 17α-valerate is for use asa Glucocorticoid Receptor (GR) modulator, preferably a glucocorticoidantagonist.

According to another aspect, there is provided a pharmaceuticalcomposition comprising a compound of the following structural formula:

and at least one physiologically acceptable excipient for use as amedicament, preferably in the treatment of precancerous lesions,dysplasias, metaplasias and tumor diseases, optionally includingmalignant neoplasias and metastasis. Preferably said tumor diseases aresolid tumors, preferably epithelial tumors, such as prostate carcinoma;mammary carcinoma; pancreatic carcinoma; lung carcinoma;gastrointestinal tract carcinoma, such as colon carcinoma; kidneycancer; thyroid carcinoma; uterine carcinoma; adrenal carcinoma.

According to another embodiment, said epithelial tumor is prostatecarcinoma. In another preferred embodiment of the invention, the tumordiseases are prostate cancer with mutated or truncated AndrogenReceptors. In this manner, the prostate cancer that may be treatedaccording to the invention may be or have become resistant toanti-androgen targeted therapy, such as enzalutamide.

According to another embodiment, the epithelial tumors are pancreaticcarcinoma, preferably exocrine pancreatic carcinoma.

According to another embodiment, the epithelial tumors are mammarycarcinoma, preferably triple negative breast cancer (TNBC). In oneembodiment, the mammary carcinoma is triple negative breast cancer andthe subject is a relapsed or a non-responder to conventional therapy.

According to another embodiment, the epithelial tumors aregastrointestinal tract carcinoma, such as colon carcinoma.

The pharmaceutical composition may also comprise at least one otheractive ingredient, preferably a chemotherapeutic active ingredient, forsimultaneous, separate or sequential administration.

According to another aspect, there is provided a pharmaceuticalcomposition comprising a compound of the following structural formula:

and at least one physiologically acceptable excipient for use as aGlucocorticoid Receptor (GR) modulator, preferably a glucocorticoidantagonist.

In another aspect, there is provided a method of treating precancerouslesions, dysplasias, metaplasias and tumor diseases in a subject in needthereof, comprising administrating a therapeutically effective amount ofa compound of the following structural formula:

or a pharmaceutical composition comprising said compound to saidsubject.

According to one embodiment, the tumor diseases are malignant neoplasiasor metastasis.

Preferably, the subject is a mammal. Ideally, the mammal is a human.

According to one embodiment, the tumor diseases are solid tumors.Optionally, the solid tumors are epithelial tumors. The epithelialtumors may be selected from prostate carcinoma, mammary carcinoma,uterine carcinoma, pancreatic carcinoma, lung carcinoma,gastro-intestinal tract carcinoma (preferably colon carcinoma), kidneycancer, thyroid carcinoma, uterine carcinoma and adrenal carcinoma andthe like.

According to another embodiment, the epithelial tumors are prostatecarcinoma, pancreatic carcinoma, exocrine pancreatic carcinoma, ormammary carcinoma.

According to another embodiment, said epithelial tumor is prostatecarcinoma. In another preferred embodiment of the invention, the tumordiseases are prostate cancer with mutated or truncated AndrogenReceptors. In this manner, the prostate cancer that may be treatedaccording to the invention may be or have become resistant toanti-androgen targeted therapy, such as enzalutamide.

According to another embodiment, the epithelial tumors are pancreaticcarcinoma, preferably exocrine pancreatic carcinoma.

According to another embodiment, wherein the mammary carcinoma is triplenegative breast cancer. In one embodiment, the mammary carcinoma istriple negative breast cancer and the subject is a relapsed or anon-responder to conventional therapy.

According to another embodiment, the epithelial tumors aregastrointestinal tract carcinoma, such as colon carcinoma.

According to another aspect of the invention, there is provided a methodof treating a disease or disorder mediated by glucocorticoid, in asubject in need thereof, the method comprising administrating atherapeutically effective amount of cortexolone 17α-valerate or apharmaceutical composition comprising cortexolone 17α-valerate.

EXAMPLES Example 1: In-Vitro Antitumor Activity of Cortexolone17α-Benzoate (CB-03-06) on Prostate Cancer Cell Lines

The experiment was performed to test and to define the antitumoractivity in vitro of cortexolone 17α-benzoate on LNCaP (AR⁺) and PC3(AR⁻), representative of Prostatic cancer cell lines with AndrogenReceptor (AR) positive or negative expression, respectively. Theexperimental method consisted of:

-   -   1. 3000 cancer cells were seeded in 96-well flat bottom plates        in complete media containing 2% charcoal stripped bovine serum.    -   2. After 24 hours, 10 nM DHT (dihydrotestosterone) with or        without anti-androgen compounds, or DMSO vehicle (negative        control) was added to the cultures.    -   3. After 3 days, viable cell numbers were quantitated using an        ATP-dependent proliferation assay.

The aim of the test was to determine the concentration at which eachcompound kills 50% of the cancer cells (IC₅₀) in view of a potentialapplication of the compound in in vivo animal test.

Data from Experiment 1 was fitted through sigmoidal dose response curvesand analyzed using Prizm statistical analysis software. Data fromExperiment 2 were analyzed using nonlinear regression least squarescurve fit in Prizm statistical analysis software.

The IC₅₀ value found for each line is reported in the following table,compared to well known comparators the most potent anti-androgenicsteroid, CPA, and Enzalutamide, an oral Androgen Receptor inhibitor ableto prolong survival in men with metastatic castration-resistant prostatecancer currently used to treat prostate cancer. The results from 2 setsof experiments follow.

Experiment 1

The results were fitted through sigmoidal dose response curves in Prizmstatistical analysis software.

IC₅₀ (microM) CB-03-06 IC₅₀ (microM) Tumour [Cortexolone CyproteroneAcetate IC₅₀ (microM) Cell lines 17α-benzoate] IC₅₀ Enzalutamide LNCaP12 29 40 PC 3 29 98 208

IC₅₀ values show that the antitumor activity of Cortexolone17α-benzoate, even though with a weak correlation trend, could beconsidered not strictly dependent on the Androgen Receptor expression,differently from the comparators.

Experiment 2

The results below include additional experiments to those inExperiment 1. The results were analyzed using nonlinear regression leastsquares curve fit in Prizm statistical analysis software.

IC₅₀ (microM) CB-03-06 IC₅₀ (microM) Tumor [Cortexolone CyproteroneAcetate IC₅₀ (microM) Cell lines 17α-benzoate] IC₅₀ Enzalutamide LNCaP12 22 38 PC 3 28 90 180

Example 2: In-Vitro Antitumor Activity of Cortexolone17α-Valerate-21-Propionate (CB-03-10) on Prostate Cancer Cell Lines

The experiment was performed to test and to define antitumor activity invitro of cortexolone 17α-valerate-21-propionate (CB-03-10) on LNCaP(AR⁺) and PC3 (AR⁺), representative of Prostatic cancer cell lines withAR positive or negative expression, respectively. The experimentalmethod consisted of:

-   -   1. 3000 cancer cells were seeded in 96-well flat bottom plates        in complete media containing 2% charcoal stripped bovine serum.    -   2. After 24 hours, 10 nM DHT (dihydrotestosterone) with or        without anti-androgen compounds, or DMSO vehicle (negative        control) was added to the cultures.    -   3. After 3 days, viable cell numbers were quantitated using an        ATP-dependent proliferation assay.

The aim of the test was to determine the concentration at which eachcompound kills 50% of the cancer cells (IC₅₀) in view of a potentialapplication of the compound in in vivo animal test.

Data from Experiment 1 was fitted through sigmoidal dose response curvesand analyzed using Prizm statistical analysis software. Data fromExperiment 2 was analyzed using nonlinear regression least squares curvefit in Prizm statistical analysis software.

The IC₅₀ value found for each line is reported in the following table,compared to well known comparators: the most potent anti-androgenicsteroid, CPA, and Enzalutamide, an oral AR antagonist able to prolongsurvival in men with metastatic castration-resistant prostate cancer.

Experiment 1

The results were fitted through sigmoidal dose response curves in Prizmstatistical analysis software.

IC₅₀ (microM) CB-03-10 [Cortexolone IC₅₀ (microM) Tumor 17α-valerate-21-Cyproterone Acetate IC₅₀ (microM) Cell lines propionate] IC₅₀Enzalutamide LNCaP 13 29 40 PC 3 55 98 208

Experiment 2

The results below include additional experiments to those inExperiment 1. The results were analyzed using nonlinear regression leastsquares curve fit in Prizm statistical analysis software.

IC₅₀ (microM) CB-03-10 [Cortexolone IC₅₀ (microM) Tumor 17α-valerate-21-Cyproterone Acetate IC₅₀ (microM) Cell lines propionate] IC₅₀Enzalutamide LNCaP 10 22 38 PC 3 50 90 180

IC₅₀ values show that the antitumor activity of cortexolone17α-valerate-21-propionate (CB-03-10) could correlate with the AndrogenReceptor expression in the cell lines.

Example 3: In-Vitro Antitumor Activity of Cortexolone 17α-Benzoate(CB-03-06) on Pancreatic Cancer Cell Lines

The experiment was performed to test and to define the antitumoractivity in vitro of cortexolone 17α-benzoate on two pancreatic tumorcell lines, Panc1 (AR⁺) and MiaPaca2 (AR low), representative ofPancreatic cancer cell lines.

The lines were also classified as positive (AR⁺) or negative/low(AR^(+/−)) for the presence and expression of the Androgen Receptor.

The experimental method consisted of:

-   -   1. 3000 cancer cells were seeded in 96-well flat bottom plates        in complete media containing 2% charcoal stripped bovine serum    -   2. After 24 hours, 10 nM DHT (dihydrotestosterone) with or        without anti-androgen compounds, or DMSO vehicle (negative        control) was added to the cultures.    -   3. After 3 days, viable cell numbers were quantitated using an        ATP-dependent proliferation assay.

The aim of the test was to determine the concentration at which eachcompound kills 50% of the cancer cells (IC₅₀) in view of a potentialapplication of the compound in in vivo animal test.

Data from Experiment 1 were fitted through sigmoidal dose responsecurves and analyzed using Prizm statistical analysis software. Data fromExperiment 2 were analyzed using nonlinear regression least squarescurve fit in Prizm statistical analysis software.

The IC₅₀ value found for each line is reported in the following table,compared to well known comparators the most potent anti-androgenicsteroid, CPA, and Enzalutamide, a potent oral AR antagonist

Experiment 1

The results were fitted through sigmoidal dose response curves in Prizmstatistical analysis software.

IC₅₀ (microM) CB-03-06 IC₅₀ (microM) Tumor [Cortexolone CyproteroneAcetate IC₅₀ (microM) Cell lines 17α-benzoate] IC₅₀ Enzalutamide Panc130 54 156 MiaPaca2 23 46 77

Experiment 2

The results below include additional experiments to those inExperiment 1. The results were analyzed using nonlinear regression leastsquares curve fit in Prizm statistical analysis software.

IC₅₀ (microM) CB-03-06 IC₅₀ (microM) Tumor [Cortexolone CyproteroneAcetate IC₅₀ (microM) Cell lines 17α-benzoate] IC₅₀ Enzalutamide Panc128 46 111 MiaPaca2 20 39 65

IC₅₀ values show that the antitumor activity of cortexolone 17α-benzoateis at least twice higher than the activity of the comparators (CPA andEnzalutamide). Since MiaPaca2 are characterized by a low/null ARexpression, the anti-cancer activity of the compound is not directlycorrelated to the Androgen Receptor expression in the cancer cell lines.

Example 4: In Vitro Antitumor Activity of Cortexolone17α-Valerate-21-Propionate (CB-03-10) on Pancreatic Cancer Cell Lines

The experiment was performed to test and to define the antitumoractivity in vitro of cortexolone 17α-valerate-21-propionate (CB-03-10)on cell lines representatives of pancreatic tumors, namely Panc1 (AR⁺)and MiaPaca2 (AR low), representative of Pancreatic cancer cell lines.

The lines were also classified as positive (AR⁺) or negative/low(AR^(+/−)) for the presence and expression of the Androgen Receptor.

The experimental method consisted of:

-   -   1. 3000 cancer cells were seeded in 96-well flat bottom plates        in complete media containing 2% charcoal stripped bovine serum    -   2. After 24 hours, 10 nM DHT (dihydrotestosterone) with or        without anti-androgen compounds, or DMSO vehicle (negative        control) was added to the cultures.    -   3. After 3 days, viable cell numbers were quantitated using an        ATP-dependent proliferation assay.

The aim of the test was to determine the concentration at which eachcompound kills 50% of the cancer cells (IC₅₀) in view of a potentialapplication of the compound in in vivo animal test.

Data from Experiment 1 were fitted through sigmoidal dose responsecurves and analyzed using Prizm statistical analysis software. Data fromExperiment 2 were analyzed using nonlinear regression least squarescurve fit in Prizm statistical analysis software.

The IC₅₀ value found for each line is reported in the following table,compared to well known comparators: the most potent anti-androgenicsteroid, CPA, and Enzalutamide, an oral AR antagonist.

Experiment 1

The results were fitted through sigmoidal dose response curves in Prizmstatistical analysis software.

IC₅₀ (microM) CB-03-10 [Cortexolone IC₅₀ (microM) Tumor 17α-valerate-21-Cyproterone Acetate IC₅₀ (microM) Cell lines propionate] IC₅₀Enzalutamide Panc1 66 54 156 MiaPaca2 43 46 77

Experiment 2

The results below include additional experiments to those inExperiment 1. The results were analyzed using nonlinear regression leastsquares curve fit in Prizm statistical analysis software.

IC₅₀ (microM) CB-03-10 IC₅₀ (microM) Tumor [Cortexolone 17α- CyproteroneAcetate IC₅₀ (microM) Cell lines valerate-21-propionate] IC₅₀Enzalutamide Panc1 60 46 111 MiaPaca2 37 39 65

IC₅₀ values show that the antitumor activity of Cortexolone17α-valerate-21-propionate (CB-03-10) is not correlated with theAndrogen Receptor expression on the pancreatic cancer cell lines.

Example 5: In-Vivo Human Pancreatic Tumor Xenograft in Mice

The activity of cortexolone 17α-benzoate (CB-03-06) on pancreaticxenograft tumor growth in nude male mice has been evaluated incomparison with the most potent anti-androgenic steroid CyproteroneAcetate (CPA).

Cortexolone 17α-benzoate and Cyproterone Acetate were separately dilutedin DMSO/2-hydroxypropyl β-cyclodextrin (vehicle).

The test was carried out comparing the anti-tumor activity ofcortexolone 17α-benzoate at two different dosages (8.0 mg/kg,corresponding approximately at 230 μM, and 40 mg/kg, correspondingapproximately at 1150 μM), versus the vehicle (i.e. 0.4% (v/v) tween 80and 0.5% (w/v) carboxymethylcellulose in normal saline) and versus thecomparator Cyproterone Acetate at two different dosages (7.4 mg/kg and37 mg/kg).

1×10⁶ MiaPaca-2 cells suspended in matrigel were subcutaneously injectedinto 6 week old athymic nude mice.

The treatment with the tested compounds, with the vehicle and with thecomparative compound, was initiated after the tumor volume has reached50 mm³ after transplantation. All compounds were injected 100 μL/mousesubcutaneously of low dose solution (approximately 230 μM) or 100μL/mouse of high dose solution (approximately 1150 μM) of cortexolone17α-benzoate, vehicle and cyproterone acetate, respectively. Compoundsand controls were administered subcutaneously daily for 28 days.

Tumors were measured every 4 days with a digital caliper.

The results are plotted in FIG. 1 as average change in tumor volumerelative to the start of treatment. Tumor volume was calculatedaccording to the formula 0.5236(r₁)²(r₂) where r₁<r₂.

Error bars are the SEM for 7 to 10 mice per treatment group. P valueswere calculated according to the Student's t test.

The high dose of Cortexolone 17α-benzoate maintained the pancreatictumor size increase of less than 5-fold of the size of the tumor whentreatment was initiated. In contrast, the average tumor in the vehicleand in the Cyproterone Acetate treatment groups increased in size to12-fold. From these data the anti-tumoral activity of the compound ofthe present invention, cortexolone 17α-benzoate, is evident.

Example 6—In-Vivo Human Pancreatic Tumor Xenograft in Mice

The activity of cortexolone 17α-valerate-21-propionate (CB-03-10) onxenograft model of pancreatic tumor in nude male mice has been evaluatedin comparison with the anti-androgenic steroid Cyproterone Acetate(CPA).

Cortexolone 17α-valerate-21-propionate (CB-03-10) and CyproteroneAcetate were separately diluted in DMSO/2-hydroxypropyl β-cyclodextrin(vehicle).

The test was carried out comparing the anti-tumor activity ofcortexolone 17α-valerate-21-propionate (CB-03-10) at two differentdosages (approximately 8.6 mg/kg and 43 mg/kg) versus the vehicle (i.e.0.4% (v/v) tween 80 and 0.5% (w/v) carboxymethylcellulose in normalsaline) and versus the comparator Cyproterone Acetate at two differentdosages (7.4 mg/kg and 37 mg/kg).

1×10⁶ MiaPaca-2 cells suspended in matrigel were subcutaneously injectedinto 6 week old athymic nude mice.

The treatment with the tested compound, with the vehicle and with thecomparative compound was initiated after the tumor has reached a volumeof 50 mm³ after implantation, injecting subcutaneously 100 μL/mouse oflow dose solution (approximately 230 μM) or 100 μL/mouse of high dosesolution (approximately 1150 μM) of cortexolone17α-valerate-21-propionate (CB-03-10), vehicle and cyproterone acetate,respectively. Compounds and controls were administered subcutaneouslydaily for 28 days.

Tumors were measured every 4 days with a digital caliper.

The results are plotted in FIG. 2 as average change in tumor volumerelative to the start of treatment. Tumor volume was calculatedaccording to the formula 0.5236(r₁)²(r₂) where r₁<r₂.

Error bars are the SEM for 7 to 10 mice per treatment group. P valueswere calculated according to the Student's t test.

The high dose of Cortexolone 17α-valerate-21-propionate (CB-03-10)maintained the pancreatic tumor size increase to less than 5-fold theinitial tumor size for the time of treatment. Moreover, when thetreatment was stopped, the tumor size tended to increase again, but witha lower rate and extent. In contrast, the average tumor in the vehicleand in the Cyproterone Acetate treatment groups increased in size to12-fold and more, bringing to the need of suppressing some of the animalof these groups for ethical reasons. From these data the antitumoractivity of the compound of the present invention, cortexolone17α-valerate-21-propionate (CB-03-10), is evident.

From the data of Examples 5 and 6, the in vivo antitumor activity ofcortexolone 17α-benzoate and cortexolone 17α-valerate-21-propionate(CB-03-10) in vivo against the pancreatic tumor was confirmed, and boththe compounds had an antitumor activity higher than Cyproterone Acetatein the same animal model (see FIG. 3).

Example 7: In-Vitro Therapeutic Index on Pancreatic Cancer Cells Lines

In order to evaluate the safety of the compounds to be tested in thecell lines viability experiments, all the factor impacting on the cellsurvival and viability should be taken into account. In this sense, theevaluation of intrinsic toxicity of compound and comparators is reallyimportant. The ratio from IC₅₀ of the compounds on peripheral bloodmononuclear cells (PBMC) and the IC₅₀ on cancer cell lines constitutethe Therapeutic Index and show what is the safer compound to be tested.

The IC₅₀ in PBMC were tested in 2 different activation status:

stimulated—actively dividing cells

resting—quiescent, non-dividing cells

Results are reported in the below tables, relevant to, respectively,stimulated PBMC and resting PBMC:

IC₅₀ (microM) on Stimulated PBMC Experiment 1

CB-03-10 CB-03-06 [Cortexolone 17α- [Cortexolone 17α- valerate-21-benzoate] propionate IC₅₀ Cyproterone Acetate Enzalutamide IC₅₀ Celllines IC₅₀ (microM) (microM) IC₅₀ (microM) (microM) Panc1 23 68 52 159MiaPaca2 17 34 39 79 PBMC 113 106 63 52

Experiment 2

CB-03-10 CB-03-06 [Cortexolone 17α- [Cortexolone 17α- valerate-21-benzoate] propionate IC₅₀ Cyproterone Acetate Enzalutamide IC₅₀ Celllines IC₅₀ (microM) (microM) IC₅₀ (microM) (microM) Panc1 28 60 46 110MiaPaca2 20 37 39 65 PBMC 97 94 62 90

In parallel the same experiments have been repeated on resting PBMCobtaining the results here below.

IC₅₀ (microM) on Resting PBMC Experiment 1

CB-03-06 CB-03-10 [Cortexolone [Cortexolone 17α- Cyproterone17α-benzoate] valerate-21-propionate Acetate Cell lines IC₅₀ (microM)IC₅₀ (microM) IC₅₀ (microM) Panc 1 23 68 52 MiaPaca2 17 34 39 PBMC 100114 18

Experiment 2

CB-03-06 [Cortexolone CB-03-10 [Cortexolone Cyproterone 17α-benzoate]17α-valerate-21- Acetate Cell lines IC₅₀ (microM) propionate IC₅₀(microM) IC₅₀ (microM) Panc1 28 60 46 MiaPaca2 20 37 39 PBMC 85 120 84

The resulting Therapeutic Index (TI) calculated on stimulated PBMC isreported in the tables below:

TI on Stimulated PBMC Experiment 1

CB-03-06 TI CB-03-10 TI [Cortexolone [Cortexolone Cyproterone 17α-17α-valerate- Acetate Enzalutamide Cell lines benzoate] 21-propionate TITI Panc1 5 2 1 0 MiaPaca2 7 3 2 1

Experiment 2

CB-03-06 TI CB-03-10 TI [Cortexolone [Cortexolone 17α- 17α-valerate-Cyproterone Enzalutamide Cell lines benzoate] 21-propionate Acetate TITI Panc1 3 2 1 1 MiaPaca2 5 3 2 1and the resulting Therapeutic Index calculated on resting PBMC isreported in the tables below:

TI on Resting PBMC Experiment 1

CB-03-06 TI CB-03-10 TI [Cortexolone 17α- [Cortexolone 17α- CyproteroneCell lines benzoate] valerate-21-propionate Acetate TI Panc1 4 2 0MiaPaca2 6 3 0

Experiment 2

CB-03-06 TI CB-03-10 TI [Cortexolone 17α- [Cortexolone 17α- CyproteroneCell lines benzoate] valerate-21-propionate Acetate TI Panc1 3 2 4MiaPaca2 4 3 1

In the tables, the 0 value indicates higher toxicity in PBMC than in thecancer cell lines

Example 8: In-Vitro Antitumor Activity of Cortexolone 17α-Benzoate andCortexolone 17α-Valerate-21-Propionate (CB-03-10) on EpithelialIntestinal Cancer Cell Lines

The experiment was performed to test and define anticancer activity invitro of Cortexolone 17α-benzoate and cortexolone17α-valerate-21-propionate on cell lines representatives of epithelialintestinal tumors, namely HT29. The experiment method consisted in:

-   -   1. Monolayer HT-29 cells were plated in: 96-wells plates at the        density of 2×10⁴ cells/mL. The cells plated were kept at 37° C.        in 5% CO₂ and left to attach for 24 h.    -   2. Thereafter the cells were incubated for 72 h with the test        compounds at the concentrations each of 0.16, 0.8, 4, 20, 100        and 500 mM.    -   3. After 72 h of treatment, the MTT colorimetric assay was        performed.

The aim of the test was to determine the concentration at which eachcompound kills 50% of the cancer cells (IC₅₀) in view of a potentialapplication of the compound in in vivo animal test.

Data were analyzed using nonlinear regression least squares curve fit inPrizm statistical analysis software.

The IC₅₀ value found for each line is reported in the following table.

Inhibition (%) at different micromolar concentration for the twoproducts on HT29

Micromolar CB-03-10 [Cortexolone 17α- CB-03-06 concentrationsvalerate-21-propionate] [Cortexolone17α-benzoate] 0.8 0.44% −1.55% 414.23% 20.40% 20 25.49% 53.60% 100 89.77% 92.24% 500 92.10% 92.31%

The IC₅₀ values calculated for the two product (reported here below)show that both compounds show an evident anticancer activity on theHT29.

IC₅₀ Calculated (Micromolar Concentration)

CB-03-06 15.97 CB-03-10 34.16

Example 9: In-Vitro Therapeutic Index on Epithelial Intestinal CancerCells Lines

In order to evaluate the safety of the compounds to be tested in thecell lines viability experiments, all the factor impacting on the cellsurvival and viability should be taken into account. In this sense, theevaluation of intrinsic toxicity of compound and comparators is reallyimportant. The ratio between IC₅₀ of the compounds on PBMC and the IC₅₀on cancer cell lines constitute the Therapeutic Index, a parameterimportant to define the product efficacy in safe conditions.

The IC₅₀ in PBMCs were tested in 2 different activation status:

Stimulated—actively dividing cells

Resting—quiescent, non-dividing cells.

The resulting Therapeutic Index (TI) calculated on Stimulated andResting PBMC are reported in the hereunder tables:

Experiment 1

CB-03-06 (TI)[Cortexolone CB-03-10 (TI)[Cortexolone Product17α-benzoate] 17α-valerate-21-propionate Stimulated 7 3 Resting 6 3

Experiment 2

CB-03-06 (TI)[Cortexolone CB-03-10 (TI)[Cortexolone Product17α-benzoate] 17α-valerate-21-propionate Stimulated 6 3 Resting 5 4

From these data the anti-tumoral activity and safety of the compound ofthe present invention, cortexolone 17α-valerate-21-propionate, wasconfirmed versus the epithelial intestinal cancer cells.

Example 10—Synthesis of Cortexolone 17α-benzoate

Cortexolone 17α-benzoate was prepared according to a synthesis schemeincluding the following steps:

In Step 1 Cortexolone was dissolved in a suitable solvent (e.g. ethylacetate). Pyridinium tosilate or p-toluene sulfonic acid was added incatalytic amount (1-10% mol) followed by tri-alkyl orthobenzoate(R=methyl or R=ethyl). The reaction mixture was heated up to 80° C. for3 to 6 hours.

After removal of the solvent and crystallization in alcoholic solvent,cortexolone orthobenzoate 1 was obtained as a solid.

In Step 2, cortexolone orthobenzoate 1 (R=methyl or R=ethyl) wasdissolved in an alcoholic solvent (e.g. methanol) and treated with 0.1Nacetic buffer at reflux. After removal of the solvent, the residue waspurified by treatment with demi water and cortexolone-17-α-benzoate wasrecovered as a solid.

Example 11—Synthesis of Cortexolone 17α-Valerate-21-Propionate(CB-03-10) (3) and Cortexolone 17α-Valerate (CB-03-05) (2)

Cortexolone 17α-valerate-21-propionate (CB-03-10) was prepared accordingto the following synthetic scheme:

Step 1: Cortexolone was dissolved in a suitable solvent (e.g. ethylacetate). Pyridinium tosilate or p-toluene sulfonic acid was added incatalytic amount (1-10% mol), followed by tri-alkyl orthovalerate(R=methyl or R=ethyl). The reaction mixture was heated up to 80° C. for3-5 hours and, after removal of the solvent and crystallization inalcoholic solvent, cortexolone orthovalerate 1 was obtained.

In Step 2, cortexolone orthovalerate 1 (R=methyl or R=ethyl) wasdissolved in an alcoholic solvent (e.g. methanol) and treated with 0.1Nacetic buffer (pH 3 to 3.9) at reflux. After the removal of the solventfollowed by treatment with purified water, cortexolone-17α-valerate 2was recovered as a solid.

In Step 3, cortexolone-17α-valerate 2 was dissolved in pyridine andadded with 1 equivalent of propionyl chloride. When the conversion wascomplete, the mixture was diluted with water, and the product 3 wasrecovered as a solid and purified by crystallization with alcohols.

Example 12—Analysis of In Vitro Anti-Cancer Activity of CortexoloneDerived Compound CB-03-10

The capability of CB-03-10 to inhibit the growth of cancer cell linesestablished in vitro was tested.

Cancer cell lines were seeded at 3000 cells in 96-well flat bottomplates in complete media containing 2% charcoal stripped bovine serum.After 24 hours the test compounds or DMSO/vehicle (0.1% finalconcentration as negative control) were added. CPA and Enzalutamide, twopotent recognized anti-androgens were used as positive control for cellcytotoxicity. After 3 days, viable cell numbers were quantitated usingan ATP-dependent cell viability assay (Promega Cell Titer Glo). In FIG.5 is shown a dose titration of the cytotoxicity activity of cortexolonederived compounds on human and pancreatic cell lines.

The determination of the concentration at which each compound kills 50%of the cancer cells (IC₅₀) was performed to express the capability ofCB-03-10 and other compounds to inhibit cancer cell growth. Eachcompound was titrated from 3 uM to 200 uM. After 3 days, viable cellnumbers were quantitated using an ATP-dependent proliferation assay.Data shown in Table I were analyzed using nonlinear regression leastsquares curve fit in Prizm statistical software.

TABLE I IC₅₀ of CB-03-10 tested in vitro in Prostate & Pancreatic CancerCell Lines Tissue Cell Line CB-03-01 CB-03-03 CB-03-04 CB-03-05 CB-03-06CB-03-10 Type Name C17 prop C17, 21 but 9dehy 17 but C17 val C17 benC17, 21 val Enzalutamide CPA Prostate LNCaP 33 16 46 32 12 10 38 22Cancer PC3 190 53 140 170 28 53 180 90 Pancreatic Panc1 490 70 340 74 2860 110 46 Cancer MiaPaca2 110 30 160 59 20 37 65 39

It is clear from the data shown in FIG. 5 and Table I that cortexolonederived compounds kills cancer cells at various concentration and IC₅₀.CB-03-10 kills prostate cancer cells (panel a) better than potentanti-androgen CPA. More importantly CB-03-10 inhibited in vitro growthof prostate cancer cell better then Enzalutamide a novel and potentanti-androgen drug used currently in clinic as first choice for androgendependent prostate cancers.

Interestingly, CB-03-10 inhibits growth of fast growth pancreatic celllines (panel b) that are known to express Androgen Receptor (AR) a verylow levels. These data suggest an independent mechanism of actionrelated to cytotoxicity then the anti-androgen activity.

Example 13—Analysis of Androgen Receptor Expression on Tested CancerCell Lines

A FACS assay was performed on prostate and pancreatic cell lines testedin Table I to better understand the relationship between AR expressionon cancer cells lines and the capability of CB-03-10 to inhibit cancercell growth.

FIG. 6 shows the level of AR expression on the tested cancer cells. Asexpected, FACS analysis AR expression in prostate and pancreatic celllines are consistent with published expression levels:LNCaP>Panc1>PC3=MiaPaca2.

To better clarify the correlation between AR and IC₅₀ Table I wasimplemented adding the AR expression of the tested cancer cell lines(Table II).

TABLE II AR expression of Prostate & Pancreatic Cancer Cell Lines andIC₅₀ of Cortexolone-derived Compounds Cell Line CB-03-01 CB-03-03CB-03-04 CB-03-05 CB-03-06 CB-03-10 AR Name C17 Prop C17, 21 but 9dehy17 but C17 val C17 ben C17, 21 val Enzalutamide CPA Expression LNCaP 3316 46 32 12 10 38 22 9 PC3 190 53 140 170 28 53 180 90 1 Panc1 490 70340 74 28 60 110 46 4 MiaPaca2 110 30 160 59 20 37 65 39 1

As expected the growth inhibition shown by potent anti-androgens CPA andEnzalutamide correlates with AR expression in prostate cancer cells. Theinhibitory activities of CB-03-10, also correlate (less strictly) withAR expression in prostate cancer cells. However, there is an inversecorrelation between AR expression and inhibitory activities in thepancreatic cancer cells. All tested compounds were more active in thelower AR expressing MiaPaca2 (AR+/−) compared to the Panc1 cells (AR+).This result hints on a possible AR-independent mechanism of action inpancreatic cancer. CB-03-10 is one of the most potent compounds in theseries. Notably, CB-03-10 is more potent than Enzalutamide in prostatecancer cell lines.

Example 14—Analysis of In Vitro Anti-Cancer Activity of CortexoloneDerived Compounds, in Particular CB-03-10, on a Larger Sample of CancerCell Lines Derived from Solid Tumors

Since the cytotoxic activity of CB-03-10 seemed to not correlate with ARexpression, a larger sample of solid tumors was tested. MCF7, a breastcancer cell line (AR^(+/−)), an additional pancreatic cell line withhigher AR expression (BxPC3) and intestinal cancer cell line (HT29)(AR⁻) were added to the previous panel. Results are depicted in TableIII.

TABLE III IC₅₀ of CB-03-10 on cancer cell lines characterized by AR andGR expression Genotype in vitro proliferation IC50 (μM) AR protein GRprotein CB-03-05 CB-03-10 expression expression Tissue Type Cell LineName C17 val C17, 21 val Enzalutamide relative to PC3 relative to LNCaPProstate LNCaP 32 10 38 9 1 Cancer PC3 170 53 180 1 2 22Rv1 18 positivebased on negative based on literature literature Pancreatic Panc1 74 60110 4 positive based on Cancer literature MiaPaca2 59 37 65 1 4 BxPC3 30127 3 positive based on literature Breast Cancer MCF7 50 28 129 1 2MDA-MB-231 not active 106 200 1 5 Colon Cancer HT29 530 14 150 1 2Healthy PBMC 120 120 nd positive based on Lymphocyte RESTING literaturePBMC 130 94 90 nd positive based on STIMULATED literature

CB-03-10 strongly inhibits cell viability of multiple cancer cell linesfrom different epithelial origin. The compound's cytotoxicity activitydoes not correlate with expression of AR. Additionally, CB-03-10 is morepotent than Enzalutamide in all cancer cell lines tested.

Example 15—Therapeutic Index of Cortexolone Derived Compounds onDifferent Cancer Cell Lines

The therapeutic index (TI) (also referred to as therapeutic window orsafety window or sometimes as therapeutic ratio) is a comparison of theamount of a therapeutic agent that causes the therapeutic effect to theamount that causes toxicity. IC₅₀ of the compounds was determined onfresh cells isolated from human blood (PBMC). The compounds toxicity wasdetermined as follow:Therapeutic Index=Safety/Potency=IC₅₀ stimulated PBMC/IC₅₀ cancer cell

The results are shown in Table IV

TABLE IV Therapeutic index of cortexolone derived compounds on a panelof cancer cell lines. CB-03-01 CB-03-03 CB-03-04 CB-03-05 CB-03-06CB-03-10 Tissue Type Cell Line Name C17 prop C17, 21 but 9dehy 17 butC17 val C17 ben C17, 21 val Enzalutamide CPA In vitro proliferation IC50(micro Molar) Prostate LNCaP 33 16 46 32 12 10 38 22 Cancer PC3 190 53140 170 28 53 180 90 Pancreatic Panc1 490 70 340 74 28 60 110 46 CancerMiaPaca2 110 30 160 59 20 37 65 39 BxPC3 28 30 127 Breast Cancer MCF7121 32 88 50 25 28 129 64 Colon Cancer HT29 51 30 10 14 150 Healthy PBMC0.1 140 360 130 97 94 90 62 Lymphocyte STIMULATED Therapeutic Index =IC50 resting PBMC/IC50 cancer cell Prostate LNCaP 0 9 8 4 8 9 2 3 CancerPC3 0 3 3 1 3 2 1 1 Pancreatic Panc1 0 2 1 2 3 2 1 1 Cancer MiaPaca2 0 52 2 5 3 1 2 BxPC3 3 3 1 Breast Cancer MCF7 0 4 4 3 4 3 1 1 Colon CancerHT29 7 4 6 3 Healthy PBMC Stim 1 1 1 1 1 1 1 1 Lymphocyte

All cortexolone derived compounds show a robust safety profile. CB-03-10shows a high therapeutic index. This reveals that CB-03-10 has a saferprofile compared to CPA and Enzalutamide.

Example 16—CB-03-10 Binding Affinity for the Androgen Receptor

The previous experiments demonstrated a strong cytotoxicity activity ofCB-03-10 on cancer cell lines derived from tumors of different origins.This cytotoxic activity did not completely correlate with theanti-Androgen Receptor expression on the tested cancer cells. Based onthis evidence assays to test the affinity of the compound to the AR weredesigned. To determine the relative binding affinities of CB-03-10 tothe wild type AR a competition assay using Polar Screen kit from lifeTechnologies was used. Briefly, the AR was added to a fluorescentandrogen ligand (Fluormone™ AL Green) to from the complex AR-LBD.Competitors displaced the fluorescent Fluormone™ AL Green ligand fromthe AR-LBD causing the fluorescent ligand to tumble rapidly during itsfluorescence lifetime, resulting in a low polarization value. Noncompetitors will not displace the fluorescent ligand from the complex,so the polarization value remains high. The shift in polarization valuein the presence of test compounds is used to determine relative affinityof test compounds for AR-LBD.

CB03-10 affinity for AR receptor was 1.1E-06 (IC₅₀ molar); in the sameassay the affinity of Dihydrotestosterone (a potent binder of ARreceptor) was 1.1E-08.

CB-03-10 binding affinity for the AR receptor when compared to DHT islow and characterizes CB-03-10 as an AR potential binder

Example 17—CB-03-10 Transcriptional Activity on the GlucocorticoidReceptor

The androgen and glucocorticoid hormones elicit divergent and oftenopposing effects in cells, tissues, and animals. A wide range ofphysiological and molecular biological evidence suggests that thereceptors that mediate these effects, the Androgen and GlucocorticoidReceptors (AR and GR, respectively), influence each other'stranscriptional activity. CB-03-10 GR antagonist and agonist activitieswere tested in an in vitro assay. Briefly, human kidney epithelial cellswere transfected with DNA construct containing GR binding sites linkedto luminescent based reporter molecule. After 24 hours, cells weretreated under antagonist or agonist modes. After an additional 24 hours,luminescence which is proportional to GR agonist transcriptionalactivity was quantitated.

Antagonist Assay was based on inhibition of luminescence induced byDexamethasone (Dex).

The antagonist activity of CB-03-10 was compared to a known GRantagonist, Mifepristone (also called RU486) as shown in FIG. 7.

Agonist Assay—was based on induction of luminescence by CB-03-10

The agonist activity of CB-03-10 was compared to a RU486 which is knownto not have agonist activity. As shown in FIG. 8.

As shown in FIG. 7, CB-03-10 is a potent antagonist (10 times less thanRU486). By contrast, CB-03-10 is essentially ineffective as GR agonistsince very high concentrations are required to induce an activity thatis 20% that of 50 nM Dex.

Example 18—CB-03-10 Induction of Apoptosis and Cell Cycle Arrest

Most of the cytotoxic anticancer drugs in current use have been shown toinduce apoptosis in susceptible cells. The fact that disparate agents,which interact with different targets, induce cell death with somecommon features suggests that cytotoxicity is determined by the abilityof the cell to engage this so-called ‘programmed’ cell death. CB-03-10was evaluated to determine if the mechanism of cytotoxicity on cancercell lines was mediated by apoptosis and cell cycle arrest. Cancer celllines were seeded in 6-well flat bottom plates. After 24 hours testcompounds or DMSO vehicle (negative control) were added. After anadditional 24 hours cells were scraped and stained with fluoresceinconjugated Annexin V and propidium iodide and analyzed by flowcytometry.

FIG. 9 shows clearly how CB-03-10 is able to induce apoptosis in apancreatic cancer cell lines. CB-03-10 induces apoptosis in a total of28% cells (early and late apoptosis) vs only 11% by the control.

Apoptosis can occur at the G1/S or G2/M transition of the cell cycle.Cells were treated with CB-03-10 for 24 hours then fixed withparaformaldehyde and stained with propidium iodide. Data in FIG. 10indicates CB-03-10 induces an S phase block at lower concentrations thena G2/M block at higher concentration. The lack of G1 block indicates noeffect on p53. The S & G2/M blocks may indicate activity on cell cyclecheck point molecules. For S phase, a possible target is the cyclindependent kinase 2 (CDK2). Gemzar and cisplatin are example drugs thatact in S phase. For G2, a possible target is CDK1.

Example 19—Analysis of Caspase Induction by CB-03-10

From previous studies was determined that CB-03-10 induces apoptosisusing Annexin V staining in MiaPaca2 cells. To better analyze thephenomenon the enzymatic activity of Caspase 8 (initiator caspase forExtrinsic Pathway) and Caspase 9 (initiator caspase for IntrinsicPathway) and of Caspases 3 and 7 (effector caspases) were measured.

For this purpose, MiaPaca2 cells were seeded in 96 well flat bottomculture plates. After 24 hours test compounds were added to cells,Gemcitabine (a known pancreatic cancer chemotherapeutic agent) and DMSOwere used as positive and negative control, respectively. After 8, 24, &48 hours incubation with test compounds, cells were lysed in buffercontaining the caspase 3/7 or 8 or 9 substrate and a stable luciferasein proprietary buffers. The lysates were transferred to white opaqueplates before measuring luminescence in a Tecan Safire instrument.Parallel plates treated identically, were used to determine viablecells. All caspase activities were corrected for the number of viablecells. Results are shown in FIG. 11.

The activities of caspases 8 and 9 (panels A and B) were induced byCB-03-10. This induction was quick, dose-related, and already evidentafter 8 hours and was as high as 7-fold increase compared to control.

Gemcitabine (a known chemotherapy agent used for pancreatic cancertreatment) also induced caspase 8 and 9 activities but with a delayedand less potent response compared to CB-03-10. The 2, 3-fold increase inCaspase 8 and 9 activity is not seen until the 48 hours mark.

Caspase 3/7 (panel C) were induced by CB-03-10 also in this case alreadyat 8 hours at and a really high level after 48 hours incubation.Interestingly CB-03-05 does not show a good profile for caspaseactivation. Gemcitabine increase in Caspase 3/7 activity is not seenuntil the 48 hours mark.

Same assay was repeated using LNCaP prostate cancer cell lines (FIG.12). In this case the positive control is Enzalutamide, a potent andnovel anti antiandrogen currently used in clinic to treat prostatecancer patients. The results are shown in FIG. 12 at 24 hours incubationwhen the caspase activities peaked.

The experiment clearly shows that CB-03-10 induced Initiator (8 and 9)and Effector (3/7) caspase activities better than Enzalutamide (used aspositive control). These results showed CB-03-10 strong induction ofcaspases activity on prostate cancer cell lines, affecting bothintrinsic and extrinsic pathways, confirming the inhibition observed onMiaPaca2 cell lines.

Example 20—CB-03-10 In Vitro Metabolism in Rat and Human Plasma

To obtain some insights on the metabolism of CB-03-10 in human and ratplasma, a specific assay was designed. Briefly the compound wasincubated at different time in human and rat plasma at 37° C. Afterincubation the samples were tested for presence of the intact compoundby liquid chromatography. The time course and concentration are shown inFIG. 13.

The results show that CB-03-10 is rapidly degraded to CB-03-05 in humanplasma and it is degraded more rapidly in rat compared to human plasma.

Example 21—Analysis of CB-03-10 In Vivo Pharmacokinetic in an AnimalModel (Mouse)

The pharmacokinetic of CB-03-10 was evaluated in plasma of mice afterintravenous (IV), subcutaneous (SC) and oral administration (PO).

Mice (3 per group) were administered with the following doses, blood wascollected at the indicated times. Plasma samples were analyzed byHPLC-MS/MS.

Group Dosing Route Blood Collection Time Point 1 iv (20 mg/kg) 10 min, 1hr, 4 hr 30 min, 2 hr, 8 hr 2 SC (40 mg/kg) 30 min, 2 hr, 8 hr 1 hr, 4hr, 24 hr 3 PO (40 mg/kg) 30 min, 2 hr, 8 hr 1 hr, 4 hr, 24 hr

CB-03-10 was undetectable in plasma even after 1 hour regardless of theadministration route. However, CB-03-10 metabolized to CB-03-05 with abody exposure of 189 (SC) and 47 (PO) hour/ng/ml (FIG. 14).

Example 22—In Vivo Testing of CB-03-10 in a Mouse Xenograft Model ofHuman Pancreatic Cancer (MiaPaca2 Cell Line)

From previous studies CB-03-10 was observed to strongly inhibit the invitro growth of MiaPaca2 pancreatic cells lines (AR^(+/−)). Aninvestigation to whether this result could be translated into an in vivoxenograft pancreatic cancer model was performed. CPA a well-knownanti-androgen was used as control. Briefly 1×10⁶ MiaPaca2 cellssuspended in matrigel were subcutaneously (SC) injected into 6 week oldmale athymic nude mice. Tumors were measured every 4 days with a digitalcaliper. Tumor volume was calculated according to the formula:0.5236(r1) 2(r2) where r1<r2. Treatment with CB-03-10 and controlscompounds was initiated after the tumor had reached 50 mm³. Compoundsdiluted in DMSO/2-hydroxypropyl b-cyclodextrin (vehicle) were injectedsubcutaneously (SC) daily (100 μL/mouse) at the concentration of 40mg/Kg daily for 28 consecutive days. FIG. 15 shows the average tumorincrease in the in vivo xenograft model after SC injection of CB-03-10when compared to the vehicle.

In FIG. 15, CB-03-10 shows a strong and significant in vivoanti-pancreatic tumor activity when compared to the controls. It alsoshows also a significant anti-tumor activity (p<0.5) when compared tovehicle only or CPA (not shown).

During the treatment period CB-03-10 maintained the pancreatic tumorsize increase to less than 5-fold relative to the initial size. Incontrast, the average tumor in the vehicle or CPA treatment groupsincreased in size to 12-fold. CB-03-10 besides inhibiting the tumorgrowth also showed a benefit in the mice survival. Importantly, 14 daysafter treatment was stopped, CB-03-10-treated mice still maintainedsignificantly smaller tumors compared to the vehicle only group (6-foldvs 14-fold, respectively)

Median survival was 70 days for mice treated with CB-03-10 compared to60 days for vehicle treated mice or 40 days with CPA. This difference issignificant with a 2 to 4 time higher risk of death in the vehicletreated group.

Example 23—In Vivo Testing of CB-03-10 Administered Orally in a MouseXenograft Human Prostate Cancer Model (LNCaP Cells)

From previous studies CB-03-10 was observed to be also effective ininhibiting in vitro the growth of LNCaP prostate cancer cells lines. Aninvestigation to whether this result could be translated into an in vivoxenograft prostate cancer model was performed. 3×10⁶ LNCaP cellssuspended in matrigel were subcutaneously injected (on the right flank)into 6 week old male athymic nude mice. Tumors were measured asdescribed above. Treatment with CB-03-10 and controls compounds wasinitiated after the tumor had reached 50 mm³. Formulations for dosingwere prepared in 15% Vitamin E-TPGS and 65% of a 0.5% w/v CMC solutionin 20 mM citrate buffer (pH 4). Oral dosing was daily (100 mg/Kg in 200μL/mouse) for 28 consecutive days. Results were plotted as averagechange in tumor volume relative to the start of treatment. FIG. 16 showsthe results obtained from the in vivo xenograft prostate cancer modelafter oral administration of CB-03-10. Enzalutamide a novel and potentanti-androgen was used as positive control.

The oral administered CB-03-10 showed a better anti-tumor activity thanEnzalutamide during the treatment period of 28 days. The tumor volumeincreased to only 2-fold with CB-03-10 and 3-fold with Enzalutamidecompared to 10-fold in the negative control group.

CB-03-10 is also more effective than Enzalutamide in maintaining a smallprostate tumor size increase after treatment was stopped (5 vs 8-foldchange). Even 6 weeks after treatment was stopped, the average tumorvolume in the CB-03-10 group was 3.6-times smaller than in the vehiclegroup.

Example 24—CB-03-10 Inhibition of In Vitro Prostate-Specific Antigen(PSA) Secretion from LNCaP Prostate Cancer Cells

Prostate-specific antigen, or PSA, is a protein produced by cells of theprostate gland. The PSA test measures the level of PSA in a man's blood.The blood level of PSA is often elevated in men with prostate cancer andit used as surrogate marker to test prostate cancer progression in humanpopulation. After the observation that CB-03-10 was able to inhibit invivo the growth of prostate cancer, the capability of the compound toinhibit in vitro PSA secretion from cancer cells was determined. LNCaPcells were seeded in 96 well flat bottom culture plates in mediacontaining charcoal stripped serum with or without 10 nM DHT. After 24hours test compounds are added to cells, using DMSO as the vehiclenegative control and Enzalutamide as the positive control. After 48hours incubation with test compounds, supernatants were harvested andtested with an Elisa assay for PSA and same cells were lysed for cellviability assessment.

As expected the pure anti-androgen, Enzalutamide, is potent atinhibiting PSA secretion with an IC₅₀<3 μM; CB-03-10 is also potent PSAinhibitor (IC₅₀ 9 μM). However Enzalutamide activity did not titrate aswell as CB-03-10. Of note, Cortexolone, the parent and final metaboliteof all tested compounds, is essentially inactive on PSA secretion (IC₅₀of 612 μM). When cell viability of these cells was tested, Enzalutamideshowed an IC₅₀ of 61 μM and CB-03-10 showed an IC₅₀ of 11 μM. Thisconfirms the strong growth inhibitory activity of both compounds.Importantly and interestingly, Cortexolone the parent and finalmetabolite of all tested compounds, inhibited LNCaP viability only atvery high concentration (IC₅₀ of 153 μM) and it is essentially inactiveas cytotoxic compound for cancer cell lines.

Example 25—Analysis of In Vitro Anti-Cancer Activity of CB-03-10 onBreast Cancer Cell Lines

Triple Negative Breast Cancer (TNBC) accounts for around 20% of newlydiagnosed invasive breast cancer. This subtype of cancer is notsupported by hormones estrogen and progesterone, nor by the presence oftoo many HER2 receptors, for this reason patients do not respond toconventional therapy (eg tamoxifen or Herceptin). Consequently, thiscancer is characterized by resistance to chemotherapy and low survivalin patients.

There is a correlation between this cancer resistance and high GRexpression (Cancer therapy 2013). There are clinical trials testing a GRantagonist (Mifepristone/RU486) in combination with chemotherapy for thetreatment of TNBC. However mifepristone clinical use is compromised dueto poly pharmacology tied to progesterone receptor (PR) antagonism. Toevaluate if CB-03-10 can be used as potential treatment for breastcancer, and in particular TNBC, a cytotoxic assays was performed usingbreast cancer cell lines characterized by various hormone receptorexpression

The breast cancer cell lines selected for this assay were:

MCF7 breast cancer cells (ER⁺PR⁺Her2⁺, GR^(+/−))

MDA-MB-231 TBNC cells (ER⁻PR⁻Her2⁻, GR⁺⁺) Before testing cell growthinhibition breast cancer cells were characterized for AR and GR receptorexpression by FACS as previously described. The data shown in FIG. 18confirms the receptor expression as indicated in the literature.

For the cytotoxic assay: cells seeded in 96 well flat bottom cultureplates in media containing charcoal stripped serum. After 24 hours testcompounds were added to the cells. DMSO was used as the vehicle negativecontrol and RU486 as the positive control. After 72 hours incubationcell were harvested, lysed and viability determined using the cell titerglow assay for viability.

Table VI shows the IC₅₀ of CB-03-10 on the above mentioned breast cancercells lines

MCF7 MDA-MB-231 (ER⁺PR⁺GR^(+/−)) (ER⁻PR⁻GR⁺⁺) RU486 Not active 435CB-03-10 28 106 CB-03-05 50 Not active

CB-03-10 is active on both breast cancer cell lines, but it seems moreactive in MCF7 cells than MDA-MB-231, perhaps hinting that GR is not theonly target of this compound. RU486, mifepristone, (GR/PR antagonist)does not affect, as expected, viability of GR^(+/−) MCF7 cells, whileinhibits, at a very low extent, the viability of TNBC GR⁺ MDA-MB-231cells to a maximum of 25% at 100 μM. Interestingly, CB-03-05 is onlyactive in MCF7, not in MDA-MB-231. It is not clear which receptor isresponsible for this differential effect because these cells aredifferent for at least 4 receptors. If not GR, then could be ER(Estrogen Receptor) (ER), PR (Progesterone Receptor) or Her2 which areexpressed in MCF7 but not MDA MDA-MB-231.

GENERAL CONCLUSION

These examples demonstrate that Cortexolone 17α-valerate-21-propionate(CB-03-10), in particular, has a superior activity beyond other knowncortexolone derived compounds. We have observed increased results bothin-vitro and in-vivo in terms of, for example:

I) general in-vitro anti-tumoral activity;

II) in-vitro anti-tumoral activity not directly correlated to ARexpression;

III) in-vitro anti-tumoral activity directly correlated to GRexpression;

IV) therapeutic index (TI); and

V) In vivo anti-tumoral activity against pancreatic and prostate tumors;

VI) It is clear from the data shown in Table I reproduced below thatcortexolone derived compounds kill cancer cells at various concentrationand IC₅₀. However, CB-03-06 and CB-03-10 show the best IC₅₀ whencompared to the other compounds in the cortexolone derived series acrosscancer cell lines of different origin. Even the metabolite CB-03-05 ofCB-03-10, show good IC50 value in LNCaP prostate cancer cells (IC50 32microM). The lower IC₅₀ depose for a stronger in vitro anti-tumoralactivity.

TABLE I IC₅₀ of Cortexolone-derived Compounds tested in Prostate &Pancreatic Cancer Cell Lines Cell Line CB-03-01 CB-03-03 CB-03-04CB-03-05 CB-03-06 CB-03-10 Name C17 prop C17, 21 but 9dehy 17 but C17val C17 ben C17, 21 val Enzalutamide CPA LNCaP 33 16 46 32 12 10 38 22PC3 190 53 140 170 28 53 180 90 Panc1 490 70 340 74 28 60 110 46MiaPaca2 110 30 160 59 20 37 65 39II) AR expression was tested on the cancer cell lines, see Table IIreproduced below.

In prostate cancer cell lines, as expected, the growth inhibition shownby potent anti-androgens like CPA and Enzalutamide correlates with theAR expression in prostate cancer cells (higher is the AR expressionbetter is the cytotoxic activity, expressed as lower IC₅₀). AlsoCB-03-04 shows an IC₅₀ of 46 when tested on LNCaP (prostate cancer cellline that express high level of Androgen Receptor) but an IC₅₀ muchhigher (135) when tested on PC3 that express low or null AR. Notably thecytotoxic activity of CB-03-06 and CB-03-10 is not evidently influencedby the Androgen Receptor expression on prostate cancer cells. CB-03-06and CB-03-10 are characterized by a very good IC₅₀ almost irrespectiveof the AR expression.

In pancreatic cancer cell lines, where the AR expression was low oralmost null CB-03-06 and CB-03-10 show a potent cytotoxic activity,higher then CPA and enzalutamide. The higher activity could be due to anadditional mechanism of action related to the binding to additionalreceptors.

TABLE II AR expression of Prostate & Pancreatic Cancer Cell Lines andIC₅₀ of Cortexolone-derived Compounds Cell Line CB-03-01 CB-03-03CB-03-04 CB-03-05 CB-03-06 CB-03-10 AR Name C17 prop C17, 21 but 9dehy17 but C17 val C17 ben C17, 21 val Enzalutamide CPA Expression LNCaP 3316 46 32 12 10 38 22 9 PC3 190 53 140 170 28 53 180 90 1 Panc1 490 70340 74 28 60 110 46 4 MiaPaca2 110 30 160 59 20 37 65 39 1III) The therapeutic index (TI) (also referred to as therapeutic window,safety window, or therapeutic ratio) is a comparison of the amount of atherapeutic agent that causes the therapeutic effect to the amount thatcauses toxicity. IC₅₀ of the compounds was determined on fresh cellsisolated from human blood (PBMC). The compound toxicity was determinedas follow:Therapeutic Index=Safety/Potency=IC₅₀ stimulated PBMC/IC₅₀ cancer cell

The results are shown in Table VII. All cortexolone derived compoundsshow a robust safe toxicity profile. However CB-03-06 and CB-03-10showed the highest therapeutic index when tested across all 7 cancercell lines tested in vitro.

TABLE VII Therapeutic index of cortexolone derived compounds on a panelof cancer cell lines Therapeutic Index = IC50 stimulated PBMC/IC50cancer cell Cell Line CB-03-01 CB-03-03 CB-03-04 CB-03-05 CB-03-06CB-03-10 Tissue Type Name C17 prop C17, 21 but 9dehy 17 but C17 val C17ben C17, 21 val Enzalutamide CPA Prostate LNCaP 0 9 8 4 8 9 2 3 CancerPC3 0 3 3 1 3 2 1 1 Pancreatic Panc1 0 2 1 2 3 2 1 1 Cancer MiaPaca2 0 52 2 5 3 1 2 BxPC3 3 3 1 Breast Cancer MCF7 0 4 4 3 4 3 1 1 Colon CancerHT29 7 4 6 3 AVERAGE 0 4 4 3 5 4 1 1IV) Triple negative breast cancer (TNBC) as shown in example 25. Thecytotoxic activity shown by CB-03-10 is particularly impressive becauseusually conventional therapeutic agents do not work on triple negativebreast cancer (TNBC) cell lines. TNBC is defined as the absence ofestrogen and progesterone receptor expression as well as ERBB2amplification. It has no response to endocrine or anti-ERBB2 therapies.Recent studies have found some potential therapeutic targets for TNBC.However, it still has a poor outcome. Taking into consideration thecytotoxic activity and the excellent safety profile of CB-03-10;CB-03-10 is a new and improved candidate for the clinical treatment ofthis cancer.The invention will now be described by the following numberedembodiments.

1. In one embodiment the invention is a compound of formula (I)

wherein R is hydrogen or C(O)—R₁, wherein R₁ is a linear alkyl chaincontaining 2 to 5 carbon atoms, and wherein R′ is a linear alkyl chaincontaining 3 to 6 carbon atoms or an optionally substituted aryl groupor an optionally substituted heteroaryl group.

2. In another embodiment the invention is a compound of formula (I)according to statement 1 wherein the optionally substituted aryl groupis phenyl.

3. In another embodiment the invention is a compound of formula (I)according to statement 1 wherein R₁ is hydrogen or CH₂CH₃, and R′ is—(CH₂)₃—CH₃ or phenyl.

4. In another embodiment the invention is a compound according tostatement 1 having formula:

5. In another embodiment the invention is a compound according tostatement 1 having formula:

6. In another embodiment the invention is a compound according to any ofstatements 1 to 5 for use as a medicament.

7. In another embodiment the invention is a compound according to any ofstatements 1 to 5 for use in the treatment of precancerous lesions,dysplasias, metaplasias and tumor diseases.

8. In another embodiment the invention is a compound for use accordingto statement 7, characterized in that said tumor disease includesmalignant neoplasias and metastasis.

9. In another embodiment the invention is a compound for use accordingto statement 8, characterized in that said tumor diseases are solidtumors, preferably epithelial tumors, such as prostate carcinoma;mammary carcinoma; pancreatic carcinoma; lung carcinoma;gastrointestinal tract carcinoma, such as colon carcinoma; kidneycancer; thyroid carcinoma; uterine carcinoma; adrenal carcinoma.

10. In another embodiment the invention is a compound for use accordingto statement 9, characterized in that said epithelial tumors areprostate carcinoma or pancreatic carcinoma, preferably exocrinepancreatic carcinoma.

11. In another embodiment the invention is a pharmaceutical compositioncomprising at least one compound of formula (I) according to any ofstatements 1 to 5, in association with at least one physiologicallyacceptable excipient.

12. In another embodiment the invention is pharmaceutical compositionsaccording to statement 11, characterized in that they are in solid or inliquid form.

13. In another embodiment the invention is pharmaceutical compositionsin solid form according to statement 12, characterized by being powders,freeze-dried powders, granules, pellets, tablets or capsules.

14. In another embodiment the invention is pharmaceutical compositionsin liquid form according to statement 12, characterized by beingsolutions, emulsions, suspensions or syrups.

15. In another embodiment the invention is a pharmaceutical compositionaccording to any of statements 11 to 14, characterized by containing atleast another active ingredient, preferably a chemotherapeutic activeingredient, as a combination for simultaneous, separate or sequentialadministration.

16. In another embodiment the invention is a pharmaceutical compositionaccording to statements 11 to 15 for use in the treatment ofprecancerous lesions, dysplasias, metaplasias and tumor diseases.

17. In another embodiment the invention is a pharmaceutical compositionfor use according to statement 16, characterized in that said tumordiseases include malignant neoplasias and metastasis.

18. In another embodiment the invention is a pharmaceutical compositionfor use according to statement 17, characterized in that said tumordiseases are solid tumors, preferably epithelial tumors, such asprostate carcinoma; mammary carcinoma; pancreatic carcinoma; lungcarcinoma; gastrointestinal tract carcinoma, such as colon carcinoma;kidney cancer; thyroid carcinoma; uterine carcinoma; adrenal carcinoma.

19. In another embodiment the invention is a pharmaceutical compositionfor use according to statement 18, characterized in that said epithelialtumors are prostate carcinoma or pancreatic carcinoma, preferablyexocrine pancreatic carcinoma.

What is claimed is:
 1. A method of treating a tumor disease in a subjectin need thereof, comprising administrating to said subject atherapeutically effective amount of a compound of formula (I):

wherein: R is C(O)—R₁; R₁ is hydrogen or a linear alkyl chain containing2 to 5 carbon atoms; R′ is a linear alkyl chain containing 3 to 6 carbonatoms or an optionally substituted aryl group or an optionallysubstituted heteroaryl group; and wherein R₁ and R′ are not the same,and wherein the tumor disease is an adenocarcinoma.
 2. The method ofclaim 1, wherein the adenocarcinoma is prostate carcinoma, pancreaticcarcinoma, exocrine pancreatic carcinoma, colon carcinoma or mammarycarcinoma.
 3. The method of claim 2, wherein the prostate carcinoma isor becomes resistant to anti-androgen targeted therapy.
 4. The method ofclaim 3, wherein the anti-androgen targeted therapy is enzalutamide. 5.The method of claim 2, wherein the mammary carcinoma is triple negativebreast cancer.
 6. The method of claim 5, wherein the subject is relapsedor a non-responder to conventional therapy.
 7. The method of claim 1,wherein: R is C(O)—R₁; R₁ is hydrogen or CH₂CH₃; R′ is —(CH₂)₃—CH₃ or aphenyl group; and wherein R₁ and R′ are not the same.
 8. The method ofclaim 7, wherein the compound is cortexolone 17α-valerate-21-propionate.9. The method of claim 1, further comprising administering at least oneother chemothereapeutic active ingredient to said subject.